Controlled Release Vaccines and Methods of Treating Brucella Diseases and Disorders

ABSTRACT

Methods and compositions for the treatment of  Brucella  induced diseases and disorders are disclosed herein. In preferred embodiments, the invention relates to vaccines. In additional embodiments, the invention relates to formulations capable of releasing said vaccines at a controlled rate of release in vivo. In further embodiments, the invention relates to modified strains of the bacteria  Brucella melitensis  and  Brucella abortus . In still further embodiments, the invention relates to compositions that do not induce clinical symptoms or splenomegaly in a subject receiving said compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation under 35 U.S.C. § 120 of pending non-provisionalapplication U.S. Ser. No. 16/251,757 filed Jan. 18, 2019, which is acontinuation application of U.S. Ser. No. 14/969,882 filed Dec. 15,2015, now issued as U.S. Pat. No. 10,220,084, which is a continuationunder 35 U.S.C. § 120 of, pending non-provisional application U.S. Ser.No. 13/269,382, filed Oct. 7, 2011, now issued as U.S. Pat. No.9,248,176, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/390,705 filed on Oct. 7, 2010 andentitled “Controlled Release Vaccines and Methods of Treating BrucellaDiseases and Disorders” the entire contents of which are incorporatedherein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract No.W81XWH-07-1-0304 from the Department of Defense (DOD), US Army MedicalResearch and Material Command, Contract Nos. 1U54AI057156-0100, R41AI068252-01A2, and T32-0200016, from the National Institutes of Health(NIH), and Contract Nos. 99-35204-7550 and 2002-38420-5806, from theUnited State Department of Agriculture. The government has certainrights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to methods and compositions forthe treatment of Brucella induced diseases and disorders. In preferredembodiments, the invention relates to vaccines. In additionalembodiments, the invention relates to formulations capable of releasingsaid live vaccines at a controlled rate of release in vivo. In furtherembodiments, the invention relates to modified strains of the bacteriaBrucella melitensis, and Brucella abortus either to reduce virulence orto provide a diagnostic marker. In still further embodiments, theinvention relates to compositions that do not induce clinical symptomsor splenomegaly in a subject receiving said compositions.

REFERENCE TO A SEQUENCE LISTING

The present application includes a Sequence Listing filed separately asrequired by 37 CFR 1.821-1.825.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with controlled release vaccines and use of Brucellastrains in vaccine manufacturing.

Andrews and Lowry have described compositions and methods for thediagnosis and prevention of B. abortus infection in U.S. PatentApplication Publication No. 2011/0177127. The invention describes amethod of detecting a Brucella abortus infection in an animal,comprising the steps of: a) obtaining a biological sample from saidanimal; and b) detecting the presence of at least one antibodyimmunologically specific for at least one Brucella abortus protein,wherein the presence of antibodies to the Brucella abortus proteinindicates a Brucella abortus infection in said animal.

At present, no human brucellosis vaccine is available even thoughBrucella species are isolated from 86 countries, with 500,000 new casesof brucellosis appearing each year throughout Latin America, theMediterranean littoral, Arabian peninsula, Africa, central Asia and theFar East (WHO, 2006); as a result, prevention of human brucellosis hasfocused upon the reduction in animal disease. The animal vaccine strainsemployed today are fortuitous isolates attenuated in ability to causeabortion due to reduced replication in reproductive tissues. Theattenuation of these mutants does not extend to reticuloendothelialdisease observed in mice and in humans, except in the case of the roughstrain RB51. The lack of genetic definition of fortuitous isolateslimits the usefulness of vaccine strains, preventing completedescription of their stability, and warrants caution when applied tohuman use as disclosed in Sangari et al (1998) Vaccine 16, 1640-5 andSchurig et al (1991) Vet. Microbiol. 28, 171-88. Of the currentlyavailable vaccine strains, only B. abortus S19 and B. melitensis Rev1have been tested in humans as provided for in Spink et al. Bull WorldHealth Organ (1962) 26, 409-19 and Spink & Thompson (1953) JAMA 153,1162-1165, which are hereby incorporated by reference. Rev1 was found tobe highly unsuitable with ⅔ of the “volunteers” exhibiting symptoms ofdisease and colonization by the organism. However, a subculture of S19referred to as 19-BA provided results that are more palatable. Only twovolunteers (12%) exhibited symptoms of disease, and the organism wasisolated from one of these volunteers. 19-BA was originally used tovaccinate at least 3 million people in the Soviet Union as described inVershilova Bull World Health Organ (1961) 24, 85-9. These investigatorsconcluded that there were more problems due to hypersensitivity than topersistence of the organism. Eight percent complained of headache andmalaise, and 2% showed signs of febrile illness. Clearly, a vaccine withthis much side effect would not be and should not be tolerated given ourcurrent state of knowledge. Given the potential threat this organismposes, there is a need to develop a better human vaccine.

One such example of a live vaccine against brucellosis is described inU.S. Pat. No. 7,541,447 issued to Ugalde et al. (2009). The Ugaldeinvention comprises a live vaccine for immunization, prophylaxis ortreatment of brucellosis comprising a bacterium modified by partial orcomplete deletion of the pgm gene, rendering the bacterium incapable ofsynthesizing a key enzyme in the metabolism of bacterial sugars. Thevaccine of the '447 patent discloses nucleotide sequence fragmentshaving the aforementioned deletion and is either lyophilized or is in apharmaceutical vehicle.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of Brucella induced diseases and disorders. In preferredembodiments, the invention relates to vaccines. In additionalembodiments, the invention relates to formulations capable of releasingsaid live vaccines at a controlled rate of release in vivo. In furtherembodiments, the invention relates to modified strains of the bacteriaBrucella melitensis, and Brucella abortus either to reduce virulence orto provide a diagnostic marker. In still further embodiments, theinvention relates to compositions that do not induce clinical symptomsand splenomegaly in a subject receiving said compositions.

The present invention in one embodiment discloses a vaccine compositioncomprising: (i) a Brucella strain comprising one or more attenuatinggene knockouts and further comprising a diagnostic gene knockout and(ii) an encapsulating agent comprising vitelline protein B capable ofreleasing the Brucella strain at a predetermined rate. In one aspect thecomposition disclosed hereinabove comprises an optional adjuvant or apharmaceutically acceptable carrier. In another aspect the encapsulatingagent is an alginate bead or a microsphere. In yet another aspect theBrucella strain is selected from the group consisting of Brucellamelitensis and Brucella abortus. In a related aspect the Brucellaabortus is a Brucella abortus S19 strain.

In another aspect the attenuating gene knockout is selected from thegroup consisting of ΔvjbR, ΔmucR, ΔmanB/A, Δasp24, ΔvirB1, ΔvirB2,ΔvirB3, ΔvirB4, ΔvirB5, ΔvirB6, ΔvirB7, ΔvirB8, ΔvirB9, ΔvirB10, andΔvirB11. In another aspect said diagnostic gene knockout comprises adifferentiation of infected animals from vaccinated animals (DIVA)mutant that includes ΔvirB12, Δbcsp31, and Δasp24. In yet another aspectthe attenuating gene knockout comprises ΔvjbR, ΔmucR, ΔvirB2, orΔmanB/A. In one aspect the vaccine further comprises a marker forserological testing, wherein the marker is DIVA mutant comprisingΔvirB12, Δbcsp31, Δasp24, or any combinations thereof. In another aspectthe vaccine comprises ΔmucR/DIVA, ΔvjbR/DIVA, ΔvirB2/DIVA, ΔmanB/A/DIVA,or any combinations thereof. In yet another aspect the strain is adouble mutant and further comprises a third mutation, wherein the thirdmutation is a marker for serological testing. In a specific aspect thedouble mutant is selected from the group consisting of a ΔvirB2/ΔmanB/Amutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant, ΔvirB2/ΔvjbR mutant,ΔvjbR/ΔmanB/A mutant, and ΔmucR/ΔmanB/A mutant. In one aspect the thirdmutation is DIVA mutant comprising ΔvirB12, Δbcsp31, Δasp24, or anycombinations thereof. In another aspect the strain comprisesΔvjbR/ΔmucR/DIVA mutant, ΔvirB2/ΔmanB/A/DIVA mutant, ΔvirB2/ΔvjbR/DIVAmutant, ΔvirB2/ΔmucR/DIVA mutant, ΔvjbR/ΔmanB/A/DIVA mutant, andΔmucR/ΔmanB/A/DIVA mutant.

In yet another aspect the composition described hereinabove furthercomprises one or more optional antibiotic markers, wherein theantibiotic marker is Kanamycin. In one aspect the vaccine is used for aprophylaxis, an amelioration of symptoms, a treatment, or anycombinations thereof against brucellosis in a human or an animalsubject. In another aspect the vaccine is administered by an oral, anintranasal, a parenteral, an intradermal, an intramuscular, anintraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal,a rectal, a vaginal, a sublingual, or a buccal route.

Another embodiment of the instant invention relates to a vaccinecomposition comprising: a single or a double mutant strain of Brucellacomprising one or more attenuating gene knockouts and further comprisinga diagnostic gene knockout, wherein the attenuating gene knockoutscomprise ΔmucR, ΔvjbR, ΔmanB/A, ΔvirB2, ΔvirB2/ΔmanB/A mutant,ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant, ΔvirB2/ΔvjbR mutant,ΔvjbR/ΔmanB/A mutant, ΔmucR/ΔmanB/A mutant, or any combinations thereof,wherein the diagnostic gene knockout comprises a differentiation ofinfected animals from vaccinated animals (DIVA) mutant that includesΔvirB12, Δbcsp31, Δasp24, or any combinations thereof; and anencapsulating agent comprising vitelline protein B capable of releasingthe Brucella strain at a predetermined rate.

In yet another embodiment the present invention provides a vaccinecomposition comprising (a) a single or double mutant strain of Brucellamelitensis and Brucella abortus strain comprising: (i) one or moreattenuating gene knockouts and further comprising a diagnostic geneknockout, wherein the attenuating gene knockouts comprise ΔmucR, ΔvjbR,ΔmanB/A, ΔvirB2, ΔvirB2/ΔmanB/A mutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucRmutant, ΔvirB2/ΔvjbR mutant, ΔvjbR/ΔmanB/A mutant, and ΔmucR/ΔmanB/Amutant, or any combinations thereof and (ii) a marker for serologicaltesting, wherein the marker is s differentiation of infected animalsfrom vaccinated animals (DIVA) mutant comprising ΔvirB12, Δbcsp31,Δasp24, or any combinations thereof and, (b) an encapsulating agentcomprising vitelline protein B capable of releasing the strain at apredetermined rate.

In yet another embodiment the present invention discloses a method forevaluating efficacy of a vaccine against brucellosis in an animalsubject comprising the steps of: i) providing a vaccine comprising: asingle or a double mutant strain of Brucella comprising one or moreattenuating gene knockouts and further comprising a diagnostic geneknockout, wherein the attenuating gene knockouts comprise ΔmucR, ΔvjbR,ΔvirB2, ΔmanB/A, ΔvirB2/ΔmanB/A mutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucRmutant, ΔvirB2/ΔvjbR mutant, ΔvjbR/ΔmanB/A mutant, ΔmucR/ΔmanB/A mutant,or any combinations thereof, wherein the diagnostic gene knockoutcomprises a differentiation of infected animals from vaccinated animals(DIVA) mutant that includes ΔvirB12, Δbcsp31, Δasp24, or anycombinations thereof; and an encapsulating agent comprising vitellineprotein B capable of releasing the Brucella strain at a predeterminedrate; ii) inoculating the animal subject with the vaccine; iii)providing one or more animal subjects suffering from brucellosis ornaturally infected with the Brucella strain; iv) performing a DIVA assaybased on a diagnostic or serological test to differentiate detection ofone or more antibodies against the one or more attenuating geneknockouts in the infected and the vaccinated animal subjects; and v)evaluating efficacy of the vaccine against brucellosis based on apresence or absence of the one or more antibodies against the one ormore attenuating gene knockouts. In one aspect of the method theBrucella strain is selected from the group consisting of Brucellamelitensis and Brucella abortus, wherein the Brucella abortus is aBrucella abortus S19 strain.

One embodiment of the present invention relates to a method forprophylaxis, amelioration of symptoms, or any combinations thereofagainst brucellosis in a human or animal subject comprising the stepsof: identifying the human or animal subject in need of the prophylaxis,amelioration of symptoms, or any combinations thereof againstbrucellosis; and administering a therapeutically effective amount of avaccine composition to the human or animal subject for the prophylaxis,amelioration of symptoms, or any combinations thereof againstbrucellosis, wherein the vaccine comprises:

(i) a Brucella strain comprising one or more attenuating gene knockoutsand further comprising a diagnostic gene knockout;(ii) an encapsulating agent comprising vitelline protein B capable ofreleasing the Brucella strain at a predetermined rate; and(iii) an optional adjuvant or a pharmaceutically acceptable carrier.

In one aspect of the method hereinabove the Brucella strain is selectedfrom the group consisting of Brucella melitensis and Brucella abortus.More specifically, the Brucella abortus is a Brucella abortus S19strain. In another aspect the attenuating gene knockout is selected fromthe group consisting of ΔvjbR, ΔmucR, ΔmanB/A, Δasp24, ΔvirB1, ΔvirB2,ΔvirB3, ΔvirB4, ΔvirB5, ΔvirB6, ΔvirB7, ΔvirB8, ΔvirB9, ΔvirB10, andΔvirB11. In another aspect diagnostic gene knockout comprises adifferentiation of infected animals from vaccinated animals (DIVA)mutant that includes ΔvirB12, Δbcsp31, and Δasp24. In yet another aspectthe attenuating gene knockout comprises ΔvjbR, ΔmucR, ΔvirB2, orΔmanB/A.

In related aspects the vaccine further comprises a marker forserological testing, wherein the marker is a DIVA mutant comprisingΔvirB12, Δbcsp31, and Δasp24 and comprises ΔmucR/DIVA, ΔvjbR/DIVA,ΔvirB2/DIVA, ΔmanB/A/DIVA, or any combinations thereof. In one aspectthe strain is a double mutant and further comprises a third mutation,wherein the third mutation is a marker for serological testing. Inanother aspect the double mutant is selected from the group consistingof a ΔvirB2/ΔmanB/A mutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant,ΔvirB2/ΔvjbR mutant, ΔvjbR/ΔmanB/A mutant, and ΔmucR/ΔmanB/A mutant. Ina specific aspect the third mutation is a DIVA mutant. In another aspectthe strain comprises ΔvjbR/ΔmucR/DIVA mutant, ΔvirB2/ΔmanB/A/DIVAmutant, ΔvirB2/ΔvjbR/DIVA mutant, ΔvirB2/ΔmucR/DIVA mutant,ΔvjbR/ΔmanB/A/DIVA mutant, and ΔmucR/ΔmanB/A/DIVA mutant.

In yet another aspect the vaccine further comprises one or more optionalantibiotic markers, wherein the antibiotic marker is Kanamycin. In oneaspect the vaccine is administered by an oral, an intranasal, aparenteral, an intradermal, an intramuscular, an intraperitoneal, anintravenous, a subcutaneous, an epidural, a mucosal, a rectal, avaginal, a sublingual, or a buccal route.

Another embodiment disclosed herein describes a method for prophylaxis,amelioration of symptoms, or any combinations thereof againstbrucellosis in a human or animal subject comprising the steps of: a)identifying the human or animal subject in need of the prophylaxis,amelioration of symptoms, or any combinations thereof againstbrucellosis and b) administering a therapeutically effective amount of avaccine composition to the human or animal subject for the prophylaxis,amelioration of symptoms, or any combinations thereof againstbrucellosis, wherein the vaccine comprises:

(i) a single or a double mutant strain of Brucella comprising one ormore attenuating gene knockouts and further comprising a diagnostic geneknockout, wherein the attenuating gene knockouts comprise ΔmucR, ΔvjbR,ΔmanB/A, ΔvirB2, ΔvirB2/ΔmanB/A mutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucRmutant, ΔvirB2/ΔvjbR mutant, ΔvjbR/ΔmanB/A mutant, ΔmucR/ΔmanB/A mutant,or any combinations thereof, wherein the diagnostic gene knockoutcomprises a differentiation of infected animals from vaccinated animals(DIVA) mutant that includes ΔvirB12, Δbcsp31, Δasp24, or anycombinations thereof;(ii) an encapsulating agent comprising vitelline protein B capable ofreleasing the Brucella strain at a predetermined rate; and(iii) an optional adjuvant or a pharmaceutically acceptable carrier.

In yet another embodiment the present invention provides a method forprophylaxis, amelioration of symptoms, or any combinations thereofagainst brucellosis in a human or animal subject comprising the stepsof: a) identifying the human or animal subject in need of theprophylaxis, amelioration of symptoms, or any combinations thereofagainst brucellosis and b) administering a therapeutically effectiveamount of a vaccine composition to the human or animal subject for theprophylaxis, amelioration of symptoms, or any combinations thereofagainst brucellosis, wherein the vaccine comprises:

(i) a single or double mutant strain of Brucella melitensis and Brucellaabortus strain comprising: one or more attenuating gene knockouts andfurther comprising a diagnostic gene knockout, wherein the attenuatinggene knockouts comprise ΔmucR, ΔvjbR, ΔmanB/A, ΔvirB2 ΔvirB2/ΔmanB/Amutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant, ΔvirB2/ΔvjbR mutant,ΔvjbR/ΔmanB/A mutant, ΔmucR/ΔmanB/A mutant, or any combinations thereofand a marker for serological testing, wherein the marker is adifferentiation of infected animals from vaccinated animals (DIVA)mutant;(ii) an encapsulating agent comprising vitelline protein B capable ofreleasing the Brucella strain at a predetermined rate, and(iii) an optional adjuvant or a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows protection of Balb/c mice vaccinated with the presentinvention against virulent challenge infection. Groups of five mice werevaccinated with 1×10⁶ colony forming units (CFU) of vaccine strain andchallenged at 12, 16 or 20 weeks post-vaccination. The spleens wereremoved one week later and virulent B. melitensis 16M coloniesenumerated. Significant difference determined using ANOVA is indicatedby either an asterisk (p<0.05) or double asterisk (p<0.01);

FIG. 2 shows a micrograph of alginate reservoir capsules measuring 200microns in diameter;

FIG. 3 shows clearance of B. melitensis vaccine strains from a mammalianspleen following inoculation of Balb/c mice with 1×10⁶ cfu as providedfor in Example I;

FIG. 4 shows recovery of B. melitensis 16M by bacteriologic culture fromspleen in BALB/c mice vaccinated with encapsulated B. melitensis vaccinecompositions of the present invention following 32 weekspost-vaccination as provided for in Example I. A single asterisk (*)represents data with p<0.05; double asterisks (**) represent data withp<0.01;

FIG. 5 shows INFγ production in BALB/c mice vaccinated with encapsulatedB. melitensis vaccine strains as described in Example I. Bluebar=measurements performed after 10 weeks post-injection (p.i.); redbar=measurements performed after 30 weeks p.i.; yellow bar=measurementsperformed two days after challenge doses were delivered;

FIG. 6 shows INFγ production by spleen cells in BALB/c mice vaccinatedwith encapsulated B. melitensis vaccine strains as provided for inExample I. Double asterisks (**) represent data with p<0.01;

FIGS. 7A and 7B show the kinetics of clearance of 16MΔmucR from mice.Forty, 6-8 week old female BALB/c mice were used to evaluate thepersistence and replication of 16MΔmucR. Mice were inoculatedintraperitoneally with either (FIG. 7A) 1×10⁶ CFU in 100 μl 16MΔmucR or(b) 1×10⁶ CFU in 100 μl of the parental strain 16M. Groups of four micewere euthanized via carbon dioxide asphyxiation at 1, 3, 5, 7 or 9 weekspost infection. At each time point, the spleens were harvested, weighed,and homogenized in 1 ml of peptone saline. Serial dilutions wereprepared, and 100 μl aliquots of each dilution (including the undilutedorgan) were plated in duplicate onto TSA plates. The levels of infectionwere expressed as the mean±of standard error of the mean (SEM) ofindividual log CFU/spleen. (FIG. 7B) The spleen weights were measuredand used to compare the mutant strain to wild type organism. Statisticalsignificance is based upon Student's T-Test comparing the deletionmutant to the wild type strain. The solid line at 0.69 logs representsthe lower limit of detection, with is >5 CFU. Splenomegaly, apathological consequence of infection with the wild type strain 16M isnot observed with the vaccine strain 16MΔmucR;

FIG. 8 shows the protection against homologous 16M intraperitonealchallenge. Groups of 5 female BALB/c mice were vaccinated with 16MΔmucRat either 1×10⁵ CFU/mouse, 1×10⁶ CFU/mouse, or unvaccinated as a naïvecontrol. 20 weeks post-vaccination all animals were challenged with6×10⁵ CFU/mouse IP. One week post-challenge, mice were euthanized viaCO₂ asphyxiation and spleens, livers, and lungs collected. Data arereported as the log₁₀ recovery of Brucella from organs. The solid lineat 0.69 logs represents the lower limit of detection, which is >5 CFU.Statistical analysis was performed by ANOVA for each organ separatelyfollowed by Tukey's honestly significant (HSD) post-test comparing allgroups to one another;

FIG. 9 shows the protection against homologous 16M aerosol challenge.Groups of 5 female BALB/c mice were vaccinated with 16MΔmucR at 1×10⁵CFU/mouse, or unvaccinated as a naïve control. 20 weeks post-vaccinationall animals were challenged with an aerosol chamber dose of 5×10⁹ CFU/mlof 16M. Four week post-challenge, mice were euthanized via CO₂asphyxiation and spleens, livers, and lungs collected. Data are reportedas the log₁₀ recovery of Brucella from organs. The solid line at 0.69logs represents the lower limit of detection, which is >5 CFU.Statistical analysis was performed by Student's T-Test comparingvaccinated to non-vaccinated mice for each organ separately;

FIGS. 10A and 10B show cross-protection against other species ofBrucella. Six to eight week-old female BALB/c mice are inoculatedintraperitoneally (IP) with a single dose (1×10⁵ CFU/mouse) of B.melitensis 16MΔVirB, 16MΔVirB/ManB/A, 16MΔvjbR. Controls include emptycapsules or PBS as a control. Twenty weeks post-vaccination, mice (n=5per group) were challenged i.p using 6×10⁵ CFU/mouse of B. melitensis16M (FIG. 10A) or B. abortus 2308 (FIG. 10B). One week post-challenge,animals were euthanized, spleens, lungs and livers harvested,homogenized in 1 ml of PBS and plated to determine total CFU/organ.Serial dilutions were performed and aliquots were plated onto TSA orFarrell's media plates. The levels of infection were expressed asmeans±SEMs of the individual log₁₀ CFU/spleen, log₁₀ CFU/liver and log₁₀CFU/lung;

FIG. 11 shows the survival of IRF-1^(−/−) knockout mice infected with1×10⁶ CFU/mouse of either 2308, S19, S19ΔvjbR, 16M or 16MΔvjbR. Miceinoculated with ΔvjbR vaccine candidates survived longer compared toeither 16M (P<0.005), 2308 (P<0.005) or S19 (P<0.005). In the presentstudy, the safety of the vaccine candidates in the Interferon regulatoryfactor (IRF^(−/−)) knockout mice. IRF-1^(−/−) mice infected with eitherwild-type Brucella melitensis 16M or the vaccine strain Brucella abortusS19, succumb to the disease within the first three weeks of infection,which is characterized by a marked granulomatous and neutrophilicinflammatory response that principally targets the spleen and liver. Incontrast, IRF-1^(−/−) mice inoculated with either the B. melitensis16MΔvjbR or B. abortus S19ΔvjbR, do not show any clinical or majorpathologic changes associated with vaccination. Additionally, when16MΔvjbR or S19ΔvjbR vaccinated mice are challenged with wild-typeBrucella melitensis 16M, the degree of colonization in multiple organsis significantly reduced, along with associated pathologic changes.These findings demonstrate the safety and protective efficacy of thevjbR mutant in an immunocompromised mouse model;

FIGS. 12A-12D shows microscopic changes observed in the spleens ofIRF-1^(−/−) mice vaccinated with S19ΔvjbR (FIG. 12C) or 16MΔvjbR (FIG.12D) and challenged 8 weeks post-vaccination with 1×10⁶ CFU/mouse ofwild-type B. melitensis 16M. Naïve but challenged mice (FIG. 12B) ornaïve (FIG. 12A) are present for comparison. Note the marked reductionin the inflammatory response in the spleen in animals that received theΔvjbR mutant;

FIGS. 13A-13D shows microscopic changes observed in the livers ofIRF-1^(−/−) mice vaccinated with S19ΔvjbR (FIG. 13C) or 16MΔvjbR (FIG.13D) and challenged 8 weeks post-vaccination with 1×10⁶ CFU/mouse ofwild-type B. melitensis 16M. Naïve but challenged mice (FIG. 13B) ornaïve (FIG. 13A) are present for comparison. Note the marked reductionin the inflammatory response in the liver in animals that received theΔvjbR mutant; and

FIGS. 14A-14I illustrate histological changes associated with 16MΔmucRvaccination followed by challenge with 16M strain. BALB/c mice werevaccinated with 16MΔmucR at 10⁵ CFU/mouse or left unvaccinated as naivecontrols. At 20 weeks post vaccination, all of the animals werechallenged with an aerosol chamber dose of 5×10⁹ CFU of 16M/ml. At 2weeks post-challenge, tissues were collected for histology. Thehistology of the nonvaccinated but challenged animals (FIGS. 14A-14C)were compared to vaccinated but challenged animals (FIGS. 14D-14F). Thelungs (FIGS. 14A and 14D), livers (FIGS. 14B and 14E), and spleens(FIGS. 14C and 14F) were compared to determine the reduction inpathology afforded by vaccination with the 16MΔmucR mutant. Naive miceare depicted (FIGS. 14G-14I) for comparison. Hematoxylin and eosinstaining was used (×10 magnification).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an,” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, “Brucella” refers to a genus of Gram-negative bacteria.They are small, non-motile, encapsulated coccobacilli. While notlimiting the scope of the present invention, Brucella is oftentransmitted by ingesting infected food, direct contact with an infectedanimal, or inhalation of aerosols.

As used herein, “splenomegaly” refers to a disease characterized by anenlargement of the spleen. While not limiting the scope of the presentinvention, symptoms of splenomegaly include but are in no way limited toabdominal pain, early satiety due to splenic encroachment or anemiarelated symptoms.

As used herein, the term “brucellosis” refers to a disease caused byingestion of milk or meat and/or contact with the bodily fluids orsecretions of animals infected with Brucella bacterial species. Whilenot limiting the scope of the present invention, symptoms of brucellosisinclude but are not limited to acute undulating fever, headache, nightsweats, fatigue, sterility and anorexia.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset of a disease or disorder.It is not intended that the present invention be limited to completeprevention. In some embodiments, the onset is delayed, or the severityof the disease or disorder is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, the present invention also contemplates treatmentthat merely reduces symptoms, improves (to some degree) and/or delaysdisease progression. It is not intended that the present invention belimited to instances wherein a disease or affliction is cured. It issufficient that symptoms are reduced.

The term “subject” as used herein refers to any mammal, preferably ahuman patient, livestock, or domestic pet. It is intended that the term“subject” encompass both human and non-human mammals, including, but notlimited to bovines, caprines, ovines, equines, porcines, felines,canines, etc., as well as humans. In preferred embodiments, the“subject” is a ruminant (e.g. bovine, etc.) or a human although it isnot intended that the present invention be limited to these groups ofanimals.

As used herein the term “immunogenically-effective amount” refers tothat amount of an immunogen required to generate an immune response(e.g. invoke a cellular response and/or the production of protectivelevels of antibodies in a host upon vaccination).

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.

The term “carrier” as used herein refers to a diluent, adjuvant,excipient or vehicle with which the active compound is administered.Such pharmaceutical vehicles can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical vehicles can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea, and the like. In addition,auxiliary, stabilizing, thickening, lubricating and coloring agents canbe used. When administered to a subject, the pharmaceutically acceptablevehicles are preferably sterile. Water can be the vehicle when theactive compound is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidvehicles, particularly for injectable solutions. Suitable pharmaceuticalvehicles also include excipients such as starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. The present compositions,if desired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents.

The present invention relates to methods and compositions for thetreatment of Brucella induced diseases and disorders. In preferredembodiments, the invention relates to vaccines. In additionalembodiments, the invention relates to formulations capable of releasingsaid live vaccines at a controlled rate of release in vivo. In furtherembodiments, the invention relates to modified strains of the bacteriaBrucella melitensis and Brucella abortus. In still further embodiments,the invention relates to compositions that do not induce splenomegaly ina subject receiving said compositions.

In preferred embodiments, the present invention relates to the treatmentand prevention of Brucellosis induced diseases and disorders. To date,Brucella species have been isolated from humans and domesticated animalsin nearly 90 countries, with an estimated 500,000 new cases each year.Moreover, Brucella species exhibit desirable characteristics for use asbioterrorism agents as described in Kaufmann et al. (1997) EmergingInfectious Diseases 3, 83-94 and Pappas et al. Cell Mol Life Sci (2006)63, 2229-36, incorporated herein by reference. Thus, vaccines exhibitingprolonged interaction with the immune system combined with the safety ofattenuated Brucella strains deliverable vaccines would be desirable.

A further embodiment of the present invention involves the use of doubleknockout, live B. melitensis mutants that are delivered orally usingmicroencapsulation-mediated controlled release compositions. Thesecompositions are storage-stable and compatible with a number ofpharmaceutical agents. At this time and following decades of testing,the only effective vaccines for the prevention of brucellosis are livingBrucella cells that stimulate the immune system through limitedinfection. Alternatives to the use of live, attenuated Brucellavaccines, including subunit vaccines and killed Brucella, have thus farproven non-efficacious. Live Brucella vaccines have been applied fordecades to prevent brucellosis in cattle and sheep, but their capacityfor direct use in humans has remained elusive. Previous reports havepostulated that use of attenuated strains appropriate for use in humansis difficult, since spontaneously derived strains retain some level ofvirulence and are genetically undefined as disclosed in Spink et al.(1962) Bulletin of the WHO 26, 409-19, incorporated herein by reference.Recent evaluation of attenuated mutants has confirmed the superiority oflong-term survival in stimulating a protective immune response, yet theadded safety of short-term survival cannot be overlooked in thedevelopment of human vaccines. The present invention combines theoptimal features of prolonged interaction with the immune system withenhanced safety of highly attenuated, single and/or double gene deletionBrucella mutants as a composition that is safe, free of side effects andefficacious in humans.

While not limiting the scope of the present invention in any way,ruminant transmission of Brucella may occur as a result of abortion ofthe fetus and/or shedding in milk. The best approach to minimize animaldisease and subsequent transmission to humans is to block transmissionvia contact with aborted material or to prevent the consumption ofcontaminated dairy products. Inhibiting replication in reproductivetissues prevents the release of massive numbers of organisms into theenvironment. For example, B. abortus S19 vaccine does not replicate inthe pregnant uterus due perhaps in part to erythritol present inelevated amounts in the pregnant uterus, although other defects mayexist as disclosed in Williams et al. (1962) British Journal ofExperimental Pathology 43, 530-537, incorporated herein by reference. B.melitensis Rev-1, a streptomycin-sensitive organism isolated followingback-selection from a streptomycin-dependent organism, is used toprotect against B. melitensis infections worldwide as provided for inAlton et al. Journal of Comparative Pathology (1967) 77, 293-300,incorporated herein by reference. Both S19 and Rev-1 provide protectionin ruminants despite long-term persistence in a percentage ofvaccinates, but retain significant virulence in humans as disclosed inSpink et al. Bull World Health Organ (1962) 26, 409-19. There are evenreports of human isolation of the recently approved vaccine strain RB51,a rough derivative used with some success in ruminants as described inPalmer et al. Am J Vet Res (1997) 58, 472-7, but with less success inother species Arenas-Gamboa et al. (2009) 45, 165-73 incorporated hereinby reference. While not limiting the present invention to any particulartheory, there is evidence that the absence of the immunodominant0-antigen prevents development of cross-reacting immune responses thatare indistinguishable from wild-type infection, but also increases thesusceptibility of this strain to complement-mediated lysis, limitingsurvival and vaccine efficacy. Splenocytes in infected animals expressmore mRNA for IL-2, IFNγ, and IL-10, and less mRNA for IL-4 thanuninfected animals, suggesting a T-helper type 1 (TH1) response asdisclosed in Zhan and Cheers. Infection and immunity (1995) 63, 720-3,incorporated in its entirety by reference. However, increases in IL-10may be counterproductive and may explain the virulence of this organismand the failure of some vaccines to stimulate a protective immuneresponse as disclosed in Svetić et al. International immunology (1993)5, 877-83, incorporated herein by reference. Subunit and killed vaccinecandidates have proven to be less effective than live, attenuatedorganisms, despite the use of adjuvants, conjugation to carriers,immunization (+/−cytokines), or alternate routes of inoculation.Attempts to develop subunit vaccines have met with limited success asdisclosed in Cassataro et al. Infect Immun (2005) 73, 8079-88 andKaushik et al. Vet Res Comm (2010) 34, 119-32, both are incorporatedhere by reference. The intracellular nature of this organism requiresthe stimulation of a cell-mediated immunity (Th1) favored by the use ofattenuated live vectors capable of stimulating this arm of the immunesystem. For this reason Brucella, like M. bovis BCG has been suggestedas a vector for the delivery of immunogens of other intracellular agentsas provided for in Surendran et al. Vet Micorbiol (2010) in press. Oneapproach to human vaccine design is the identification of mutants ofreduced persistence (survival) within macrophages based on therelationship to disease and potential for latent survival as disclosedin Hong et al. (2000) Infection and Immunity 68, 4102-4107, incorporatedherein by reference. To date, functions targeted for inactivation arebased on observations with other bacterial pathogens that typicallycause acute infections. The chronic nature of brucellosis in humanssuggests another approach is warranted. For this reason, the presentinventors have identified gene functions necessary for persistencewithin macrophages using signature-tagged mutagenesis as disclosed inLestrate et al. Mol Microbiol (2000) 38, 543-51, Foulongne et al. InfectImmun (2000) 68, 1297-303 and Hong et al. (2000) Infect Immun 68,4102-4107, incorporated herein by reference. In this approach, genesrequired for survival have been classified into two groups. Group I genefunctions are required for early survival in mice and are reduced atboth two weeks and eight weeks post infection. A subgroup of thesemutants recovers to normal levels by eight weeks suggesting that itsfunction is only transiently required. Group II gene functions arerequired for long-term survival appearing normal at two weeks, butreduced after eight weeks. Although persistence in animal species may beacceptable, assuming the organism does not colonize tissues thatthreaten other animals in the herd or humans, use in humans requiresmuch greater attenuations. Examination of the vaccine potential of thesegroups of organisms in the mouse model is performed here to provideproof of principle prior to testing in other species including non-humanprimates.

In some embodiments, the present invention is administered to humanbeings for the treatment and/or prevention of brucellosis. The hallmarksof human brucellosis are persistent undulating severe fevers coupledwith a measurable splenomegaly with increased lymphohistiocytic cells inthe spleen, a reduced percentage of splenic CD4+ and CD8+ T cells, andan increased percentage of splenic macrophages. Brucellosis occurs in atleast 90 countries in humans or animals, which is often difficult torecognize, and may present as an acute fever, or as a chronic orlocalized infection. Infection is best diagnosed by growing thebacterium from blood or other infected tissues. Due to the slow growthof Brucella, cultures may require several weeks for positiveidentification. Infection is also diagnosed by detection ofanti-Brucella specific antibodies in patients' blood. Humans areinfected through the ingestion of contaminated animal products, and arenormally dead-end hosts, although anecdotal evidence suggests occasionalsexual transmission as disclosed in Meltzer et al. Clin Infect Dis.(2010) 51, e12-5, incorporated herein by reference. Persistence withinthe cells and tissues of the host is the basis for human disease and thecause of reduced efficacy of antibiotic treatment. Recommended treatmentrequires a combination of deoxycycline and gentamicin or rifampin asdisclosed in Roushan et al. J Antimicrobi Chemother (2010) 65, 1028-35,hereby incorporated by reference. Disease may begin abruptly orgradually from three days to several months after exposure. Nonspecificsymptoms typically observed in humans include pyrexia, diaphoresis,fatigue, loss of appetite, and muscle or joint pain. Depression,cephalalgia, and irritability also frequently occur. Infection of bonesor joints occurs in about 1 in 3 patients, causing localizedinflammation and edema. Some may also have cough, chest pain, andstomach upset. Although about 1 in 4 patients with brucellosis haverespiratory symptoms, thoracic radiographs usually appear normal asdisclosed in Madkour M M (1989) in Brucellosis, pp. 131-139. Lubani, etal (1989) Quart J Med 71, 319-324., both of which are incorporated byreference. Less common, but of added concern are infection of the,brain, heart valves, or male reproductive system. Symptoms often lastfor 3-6 months, but occasionally persist for a year or longer and mayreappear after periods of quiescence. Chronically infected patientsfrequently experience weight loss, and many patients temporarily improvebut relapse as provided for in Ariza, et al (1986) Antimicrob. AgentsChemother. 30, 958-960., incorporated herein by reference. Brucellosiscan be treated with antibiotics (usually doxycycline and rifampin) takenorally, but requires treatment for at least six weeks. Recurrence ofhuman brucellosis may be related to a latent form of survivaldemonstrated to occur at low frequency as provided for in Ray et al(1988) J. Am. Vet. Med. Assoc. 192, 182-186, incorporated herein byreference.

Brucella organisms can be delivered via aerosol to infect humans. Theuse of Brucella as a weapon was calculated to pose a substantialfinancial risk as disclosed in Kaufmann et al. (1997) EmergingInfectious Diseases 3, 83-94 and Pappas et al. Cell Mol Life Sci (2006)63, 2229-36, both incorporated herein by reference. Infectionincapacitates human hosts with mostly flu-like symptoms, but will resultin death if left untreated as provided for in Young E J (1995) Clin InfDis 21, 283-290, hereby incorporated by reference. Bioengineering posesthe additional risk of introducing antibiotic resistance, renderingineffective the most successful form of treatment. The transposon Tn10encoding tetracycline resistance has been used to obtain stabletransformants. The financial impact study did not attempt to determinethe threshold at which financial risk may pose a risk to nationalsecurity. Nor did the study outline scenarios in which the use of oneorganism might be favored over the use of others. The study didunderscore the need to invest in research in all understudied organismsto prevent their use in this manner and suggested that decreased studyof these organisms increase the potential consequences resulting fromtheir use as weapons. Brucella spp. have been weaponized by severalcountries, including the former Soviet Union, Japan and the USA, andthus is a recognized biological warfare threat that can cause illnessand death in humans. No vaccine for humans is available against thisthreat. Expected market and commercial need: The primary need for humanbrucellosis vaccines is for specialty protection of military personnel,public health workers and veterinarians with the cross-over opportunityfor extensive markets in the high risk zones that occur throughout theworld, particularly in the Middle East, Central Asia, Latin-America,Africa and the Far East. While there is a huge need for a humanbrucellosis vaccine worldwide, the question is whether or not majorbiologics manufacturers will recognize these needs as a profitablemarket, thus it is more plausible that federal government subsidizedstockpiles, e.g., Bioshield I and Bioshield II, to protect the generalpublic and military personnel represent a more likely market. Thepotential reluctance of the general population to use live vaccines isbased on a limited trust of scientists and government, and such thinkingmust not be used to deter the development of products based on otherwisesound scientific principles. The use of such vaccines in humans isexpected under extreme circumstances, such as stockpiling large reservesfor protection against biological terrorism or biological warfare.Starting with the work of Louis Pasteur, live vaccines have offered thebest possible solution for immune protection. Use in humans requiresthat safety be determined beyond a shadow of a doubt. This is one of thereasons that the present inventors use of double knockout mutants.Questions concerning the preliminary production under Good ManufacturingProcedures and safety testing of such products warrant studies innon-human primate models.

As previously mentioned, no federally approved or commercially availablehuman brucellosis vaccines are available anywhere worldwide; theresimply are no currently known or published existing vaccine alternativesto protect humans from Brucella.

The present invention provides for controlled release compositionsfurther comprising attenuated, live Brucella mutant vaccines. Drugdelivery materials have historically been derived from many sourcesincluding commodity plastics and textile industries and have beenincorporated into vehicles as diverse as pH responsive hydrogels andpolymer microparticles or implants designed for surface or bulk erosionas disclosed in Langer RaP, N. A. (2003) Bioengineering, Food andNatural Products 49, 2990-3006, incorporated herein by reference. In thecase of controlled release formulations, a drug is typically released bydiffusion, erosion or solvent activation and transport. In most cases,the desired polymer characteristics include biocompatibility, lack ofimmunogenicity, capability of breakdown by the body and watersolubility. Many of the processes used to entrap pharmaceuticals involveharsh organic solvents which are bacteriocidal and capable of denaturingproteins. When considering controlled release vehicles for theentrapment of active enzymes or living cells, new alternatives areneeded. A number of milder processes based on established technologiesand variations have recently been applied to the delivery of activeprotein agents such as insulin, erythropoietins and chemokines asprovided for in Marschutz et al (2000) Biomaterials 21, 1499-07.Takenaga et al (2002) J Control Release 79, 81-91. and Qiu et al (2003)Biomaterials 24, 11-18., all of which are incorporated by reference, oras encapsulants for living cells to permit transplantation as disclosedin Young et al (2002) Biomaterials 23, 3495-3501, hereby incorporated byreference. The technologies cover a wide range of materials includinggelatin-based hydrogels, protein-PEG microparticles, novel PEGcopolymers, biodegradable PLGA particles, PLG/PVA microspheres andsurface modified nanospheres. Alginate, a naturally occurringbiopolymer, is especially well suited to the entrapment of living cells.Alginate is a linear unbranched polysaccharide composed of 1-4′-linkedβ-D-mannuronic acid and α-L-guluronic acids in varying quantities.Alginate polymers are highly water-soluble and easily crosslinked usingdivalent cations such as Ca2+ or polycations such as poly-L-lysine asprovided for in Wee & Gombotz (1998) Adv Drug Deliv Rev 31, 267-285,hereby incorporated by reference. The relatively mild conditionsrequired to produce either an alginate matrix or particle is compatiblewith cell viability. Entrapment in alginate has been shown to greatlyenhance viability and storage as provided for in Cui et al (2000) Int JPharm 210, 51-59 and Kwok et al (1989) Proc. Int. Symp. Contol. ReleaseBioact. Mater. 16, 170-171, both of which are incorporated by reference.The physical properties such as porosity, rate of erosion, and releaseproperties may be modulated through mixing alginates of differentguluronic acid composition and through applying different coatings tothe matrix as provided for in Wee & Gombotz (1998) Adv Drug Deliv Rev31, 267-285. While in no way limiting the scope of the presentinvention, it is generally thought that release of a biomolecule fromalginate matrices generally occurs through i) diffusion through pores ofthe polymer or ii) erosion of the polymer network. In general, thealginate matrix is stabilized under acidic conditions, but erodes slowlyat pH of 6.8 or above.

The present invention exploits the performance and safety of liveBrucella strains stabilized in an alginate bead for slow erosion of thecapsule, resulting in a prolonged release of bacteria. A furtheradvantage of the present invention is that highly attenuated, safe,double gene deletion, live Brucella mutants can be safely deliveredorally by controlled release to optimally provide the long termimmunostimulation required for protective immunity. As previouslymentioned, currently available Brucella vaccines are unsuitable forhuman use, and antibiotic therapies are at best unreliable andineffective, particularly if, e.g., bioterrorists introduce antibioticresistance into weaponized strains of B. melitensis. Vaccination offersthe best approach for long-range protection. In view of the lack ofsuccess of defining Brucella protective immunogens over the last fourdecades, the use of attenuated vaccine strains offers the best approach.Data reported below includes the identification of genetic defects thatspecifically attenuate intracellular survival. These strains have beencharacterized based on safety and attenuation in the mouse and goats andsoon in non-human primates. The most protective strains are lessattenuated, survive longer in the host and cause unwanted side effects(e.g., splenomegaly). The aim of the proposed studies is to utilize themost attenuated mutants and enhance vaccine potential through sustainedrelease ultimately in non-human primates, to list a very specific anddefined product. However, if successful the approach would provide proofof principle regarding the use of sustained delivery with liveattenuated agents by providing direct evidence by i) potentiating theefficacy of weakly persistent strains, and ii) testing the persistenceand safety of vaccine strains under conditions of controlled release forsafety of controlled release vaccines and efficacy in mouse model aswell as orally vaccinating with encapsulated mutant strains andchallenged through the aerosol route. There is strong promise for oralvaccination with alginate and alginate/protein encapsulated strains asdisclosed in Arenas-Gamboa et al. Infect Immun (2008) vol. 76, 2448-55,Kahl-McDonagh et al (2007) Infect Immun 75, 4923-32, Suckow et al (2002)J Control Release 85, 227-235, Kim et al (2002) J Control Release 85,191-202., all of which are hereby incorporated by reference. Inaddition, lyophilization of bacteria in alginate beads extends theirviability. Embodiments of the present invention include a storage-stabledelivery system that may be administered orally and is generallyapplicable to a number of select agents.

In preferred embodiments, capsule formulations include vitelline proteinB (vpB), a slowly erodable, non-antigenic protein, which extends thetime frame over which capsule dissolution occurs. The present inventionutilizes the ability of naturally occurring protein polymers (vpB) toact as controlled release vehicles. The proteins utilized for thispurpose are encapsulants produced in nature that are unusuallyrefractive to the actions of proteases, strong acids and bases. In someembodiments, alginate is formulated into the present invention.Alginate, a naturally occurring biopolymer, is especially well suited tothe entrapment of living cells. Alginate is a linear unbranchedpolysaccharide composed of 1-4′-linked β-D-mannuronic acid andα-L-guluronic acids in varying quantities. Alginate polymers are highlywater-soluble and easily crosslinked using divalent cations such as Ca²⁺or polycations such as poly-L-lysine as disclosed in Wee & Gombotz(1998) Adv Drug Deliv Rev 31, 267-285, incorporated herein by reference.The relatively mild conditions required to produce an alginate matrix orparticle are compatible with cell viability; and in many cases,entrapment in alginate has been shown to greatly enhance viability andstorage as disclosed in Cui et al (2000) Int J Pharm 210, 51-59. andKwok et al (1989) Proc. Int. Symp. Contol. Release Bioact. Mater. 16,170-171, incorporated herein by reference. Release from alginatematrices generally occurs through i) diffusion through pores of thepolymer or ii) erosion of the polymer network. In the case of standardbacterial entrapment methods used for BCG or for bifidobacteria asprovided for in Cui et al (2000) Int J Pharm 210, 51-59., cells escapethrough erosion rather than diffusion due to size and surface charge. Ingeneral, the alginate matrix is stabilized under acidic conditions, buterodes slowly at pH of 6.8 or above. Capsule formulations additionallyinclude vpB, a slowly erodable, non-antigenic protein, which extends thetime frame over which capsule dissolution occurs. Ongoing research hasidentified Brucella genes required at different stages of infection.Extensive vaccine trials in laboratory species have revealed thatinactivation of “early” genes, important early in infection, results inrapid clearance of the organism. The use of live, attenuated mutants inwhich “early” genes are inactivated or deleted, is favored, dueprimarily to the absence of side effects associated with long-termcarriage of the organism. Work with “late” mutants, identified by theirimportance late in infection has confirmed that persistence of vaccinestrains is associated with improved protective immunity. However,accompanying this increased efficacy is an increased risk of sideeffects. In order to avoid the rapid clearance of the early mutants orthe side effects associated with late mutants, the present inventors usemicroencapsulation to provide a controlled release and enhanced immuneresponse to mutants that survive for brief periods. Although otherapproaches are possible, the work proposed offers an opportunity tobuild upon historical use of attenuated live vaccine strains to protectagainst Brucella infections. Alternative sources of protection,including subunit or killed vaccine preparations have provided littlepromise for success. The development of the proposed product takes fulladvantage of ongoing research using signature-tagged mutagenesis andspecific knock-out mutants that has identified genes required forsurvival coupled to novel microencapsulation and nanoencapsulationtechnology to balance attenuation, persistence and protectiveimmunogenicity.

In one embodiment the present invention describes preparation of B.melitensis loaded alginate microspheres. Alginate beads were prepared byresuspending 6×10⁷ CFU of the live B. melitensis mutant in 1 ml of3-[N-morpholino] propanesulfonic acid] (MOPS) buffer comprising 10 mMMOPS, 0.85% NaCl, pH 7.4 and is mixed with 5 ml of alginate solution(1.5% sodium alginate, 10 mM MOPS, 0.85% NaCl, pH 7.3). Spheres wereobtained by extruding the suspension through a 1.2 cm tip into a 100 mMcalcium chloride solution and stirred for 15 min by using a NiscoEncapsulator. After extrusion of the bacterium-alginate mixture into theCaCl₂, the capsules were washed twice with MOPS for 5 min and furthercross-linked with 0.05% poly-L-lysine (molecular weight. 22,000; Sigma)and 2.5 mg of VpB for 10 min. After two successive washes, the beadswere stirred in a solution of 0.03% (wt/vol) alginate for 5 min to applya final outer shell and washed twice with MOPS before storage at 4° C.

Pharmaceutical Formulations: The present compositions can take the formof solutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. In one embodiment, the pharmaceuticallyacceptable vehicle is a capsule (see e.g., U.S. Pat. No. 5,698,155). Inone embodiment, the vaccine is encapsulated using materials described inU.S. Patent Application Publication No. 2005/0260258, herebyincorporated by reference.

In a preferred embodiment, the active compound and optionally anothertherapeutic or prophylactic agent are formulated in accordance withroutine procedures as pharmaceutical compositions adapted foradministration to human beings. Typically, the active compounds foradministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the compositions can also include a solubilizing agent.Compositions for administration can optionally include a localanesthetic such as lignocaine to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachet indicating the quantity of active agent. Where theactive compound is to be administered by infusion, it can be dispensed,for example, with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the active compound is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optional agents, for example, sweetening agents suchas fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for an oral administration of theactive compound. In these later platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving compound, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time delay material such as glycerol monostearate orglycerol stearate can also be used. Oral compositions can includestandard vehicles such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Suchvehicles are preferably of pharmaceutical grade.

Further, the effect of the active compound can be delayed or prolongedby proper formulation. For example, a slowly soluble pellet of theactive compound can be prepared and incorporated in a tablet or capsule.The technique can be improved by making pellets of several differentdissolution rates and filling capsules with a mixture of the pellets.Tablets or capsules can be coated with a film that resists dissolutionfor a predictable period of time. Even the parenteral preparations canbe made long acting, by dissolving or suspending the compound in oily oremulsified vehicles, which allow it to disperse only slowly in theserum.

Compositions for use in accordance with the present invention can beformulated in conventional manner using one or more physiologicallyacceptable carriers or excipients.

Thus, the compound and optionally another therapeutic or prophylacticagent and their physiologically acceptable salts and solvates can beformulated into pharmaceutical compositions for administration byinhalation or insufflation (either through the mouth or the nose) ororal, parenteral or mucosal (such as buccal, vaginal, rectal,sublingual) administration. In some embodiments, the administration isophthalmic (e.g. eyes drops applied directly to the eye). In oneembodiment, local or systemic parenteral administration is used.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletscan be coated by methods well known in the art. Liquid preparations fororal administration can take the form of, for example, solutions, syrupsor suspensions, or they can be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound. The microencapsulated vaccinegives a controlled release or continual boosting effect. Thoseformulations with vpB and alginate are described in U.S. PatentApplication Publication No. 2005/0260258, hereby incorporated byreference.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compositions can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The pharmaceuticalcompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the pharmaceutical compositions can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

In certain preferred embodiments, the pack or dispenser contains one ormore unit dosage forms containing no more than the recommended dosageformulation as determined in the Physician's Desk Reference (62^(nd) ed.2008, herein incorporated by reference in its entirety).

Methods of administering the active compound and optionally anothertherapeutic or prophylactic agent include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal, rectal, vaginal, sublingual, buccal or oral routes).In a specific embodiment, the active compound and optionally otherprophylactic or therapeutic agents are administered intramuscularly,intravenously, or subcutaneously. The active compound and optionallyother prophylactic or therapeutic agents can also be administered byinfusion or bolus injection and can be administered together with otherbiologically active agents. Administration can be local or systemic. Theactive compound and optionally the prophylactic or therapeutic agent andtheir physiologically acceptable salts and solvates can also beadministered by inhalation or insufflation (either through the mouth orthe nose). In a preferred embodiment, local or systemic parenteraladministration is used.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the active compound can be formulated as asuppository, with traditional binders and vehicles such astriglycerides.

Selection of a particular effective dose can be determined (e.g., viaclinical trials) by a skilled artisan based upon the consideration ofseveral factors, which will be known to one skilled in the art. Suchfactors include the disease to be treated or prevented, the symptomsinvolved, the subject's body mass, the subject's immune status and otherfactors known by the skilled artisan.

The dose of the active compound to be administered to a subject, such asa human, is rather widely variable and can be subject to independentjudgment. It is often practical to administer the daily dose at varioushours of the day. However, in any given case, the amount administeredwill depend on such factors as the viability of the active component,the formulation used, subject condition (such as weight), and/or theroute of administration.

Method of creating knockout mutants: The starting strain already has oneof the mutations ΔvjbR, ΔmucR, ΔvirB2, ΔvirB2/ΔManB/A. The method hasbeen previously described (25). The DIVA mutations include Δasp24,Δbcsp31, ΔvirB12.

Recombinant plasmid construction: In order to construct vectors toeliminate genes of interest, primers were designed to amplify sequencesflanking the genes. These flanking regions are referred to as the 5′ andthe 3′ fragments and were joined to one another using specially designedPCR primers (Table 1). Importantly, the reverse primer of the 5′fragment and the forward primer of the 3′ fragment include approximately5 to 10 nucleotides of sequence complementary to the opposite fragmentand a terminal restriction site. The 5′ and 3′ fragments were amplifiedin separate reactions, gel purified, and used as templates for a secondround of PCR (38). The forward primer of the 5′ fragment and the reverseprimer of the 3′ fragment were utilized in a second round of PCR toengineer a product that represented the ligation of the 5′ and 3′fragments. The ends of this joined product were removed by restrictiondigestion at sites engineered into the primers. The final fragment wasgel purified and ligated to pBluescript II KS(+) (Stratagene). Akanamycin cassette was inserted between the 5′ and 3′ fragmentsfollowing amplification via PCR from the plasmid pKD4 using primerscontaining the compatible restriction site located within the overlapbetween the fragments. pKD4 contains nptII from Tn5 flanked by FLPrecombination target sites (11). These constructs are referred to as themarked plasmids (Table 2). The construction of the plasmid for creationof unmarked deletion mutants entails cloning of the original overlappingPCR product (lacking the kanamycin cassette) into plasmid pEX18Ap, whichcontains sacB, using the appropriate restriction enzymes (23). Thisconstruct is referred to here as the unmarked plasmid (Table 2) (25).

TABLE 1 Primers used in this study. Primer Sequence name(restriction enzyme engineered) Fragment TAF1015′-GGAATTCGGCAAAGCGAGTGGGTGATTAG- asp24 upstream3′(EcoRI) (SEQ ID NO: 1) TAF102 5′-CGGGATCCTGAGCAAGTGCGGGAATAGC-asp24 upstream 3′(BamHI) (SEQ ID NO: 2) TAF1035′-CGGGATCCTGGGAATGGAGCGGCTTTAG- asp24 downstream3′(BamHI) (SEQ ID NO: 3) TAF104 5′-GCTCTAGATTTGAACACTTGGCGATAGCG-3′asp24 downstream (XbaI) (SEQ ID NO: 4) TAF3005′-CGGGATCCCGCACGTCTTGAGCGATTGTGTAGG- Kan cassette3′(BamHI) (SEQ ID NO: 5) TAF301 5′-CGGGATCCCGGGACAACAAGCCAGGGATGTAAC-Kan cassette 3′(BamHI) (SEQ ID NO: 6) TAF3565′-CGGGATCCCTGGAGGAAAACAATCTGGG-3′ manB/A upstream(BamHI) (SEQ ID NO: 7) TAF357 5_-AAGACGGCGCGCCCGAACCTGTATCTGCCTG-manB/A upstream 3_(AscI) (SEQ ID NO: 8) TAF358 5_- manB/A downstreamGTTCGGGCGCGCCGTCTTAACCCAAAACCGCTTCGTA- 3_(AscI) (SEQ ID NO: 9) TAF3595_-GCTCTAGAGGGTTTTCTGATCGATCTGGTAGC- manB/A downstream3_(XbaI) (SEQ ID NO: 10) TAF204 5_-GGCGCGCCACGTCTTGAGCGATTGTGTAGG-Kan cassette 3_(AscI) (SEQ ID NO: 11) TAF2055_-GGCGCGCCGGACAACAAGCCAGGGATGTAAC- Kan cassette3_(AscI) (SEQ ID NO: 12)

TABLE 2 Bacterial strains and plasmids. Strain or plasmid Relevantcharacteristic(s) B. abortus strains 2308 Wild type Strain 19 Vaccinestrain BAΔasp24::kan Δasp24::Km BAΔasp24 Δasp24 BAΔvirB2::kan ΔvirB2::Km(polar) BAΔvirB2 ΔvirB2 (nonpolar) BAΔmanBA ΔmanBA B. melitensis strains16M Wild type Rev 1 Vaccine strain BMΔasp24::kan Δasp24::Km BMΔasp24Δasp24 BMΔvirB2::kan ΔvirB2::Km (polar) BMΔvirB2 ΔvirB2 (nonpolar)BMΔmanBA::kan ΔmanBA::Km BMΔmanBA ΔmanBA E. coli strains DH5αF-ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r_(k) ⁻m_(k) ⁺)phoA supE44 λ thi-1 gyrA96 relA1 Top10 F- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araΔ 139 Δ(ara-leu)7697 galU galK rpsL(Str^(r)) endA1 nupG DH10B F-mcrA Δ(mrr-hsdRMS-mcrBC) ϕ 80lacZΔM15ΔlacX74 recA1 endA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (Str^(r)) nupGPlasmids pBluescript KS ColE1, bla pKD4 FLP/FRT, Km^(r) pEX18Ap sacB blapMMKB TAF101/TAF104 cloned into pEX18Ap pMMK8 TAF101/TAF104 cloned intopBluescipt pMMK16 pMMK8 separated by TAF300/TAF301 (kanamycinresistance) pMMK29 TAF356/TAF359 cloned into pBluescript pMMK31TAF356/TAFF359 cloned into pEX18Ap pMMK33 pMMK29 separated by TAF204/205kanamycin resistance gene pAV2.2 Plasmid to make marked virB2 deletionpAS1.1 Plasmid to make unmarked virB2 deletion

Selection of marked deletion mutants: Marked deletion mutants werecreated in B. melitensis and B. abortus via allelic exchange followingelectroporation of the marked plasmid into either 16M or 52308,respectively. Bacteria were grown as described above and pelleted viacentrifugation at 1,700×g for 15 min at 4° C. All subsequent steps wereperformed on ice or at 4° C. The cell pellet was washed three times withice-cold sterile water under the same conditions. After the final wash,the cells were resuspended in 1 ml sterile water. The bacterial cellsuspension was used in each electroporation with approximately 1 g DNAin a prechilled 1-mm gap cuvette (Bio-Rad, California) and shocked in aBTX electroporation apparatus set at 2.2 to 2.5 kV and 246. SOC-B (6%[wt/vol] tryptic soy broth, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, and 20 mM glucose) medium was immediately added to the cuvette,transferred to microcentrifuge tubes, and incubated overnight at 37° C.with agitation (31). Following incubation, the entire culture was platedonto TSA containing kanamycin. Colonies were replica plated onto TSAcontaining kanamycin and onto plates containing carbenicillin. Markeddeletion mutants from allelic exchange should be kanamycin resistant(Kmr) and carbenicillin sensitive (Carbs). Verification of mutantgenotypes was obtained via PCR and Southern blot analysis to ensure thatthe gene of interest was deleted and the kanamycin cassette wasretained.

Selection of unmarked deletion mutants. The unmarked plasmid, containingthe sacB gene, ligated 5′ and 3′ fragments, and bla gene, was used forelectroporation into marked deletion strains. Electroporation conditionswere identical to those described for the construction of markedmutants. Following electroporation, cells were plated onto TSAcontaining carbenicillin to select for the first homologousrecombination, i.e., a cointegration. Colonies were replica plated ontosucrose plates (TSA without salt, containing 6% [wt/vol] sucrose,without antibiotic) and to TSA containing carbenicillin. Colonies thatgrew on carbenicillin (Carbr) but not sucrose (Sucs) were cointegrateswith a functional sacB gene. Resolution of cointegration occursspontaneously and was selected for by inoculating 5 ml of sucrose broth(TSB, without salt or antibiotics, and supplemented with 6% [wt/vol]sucrose) and incubating for 24 h with agitation at 37° C., withsubsequent plating onto sucrose-containing medium. All knockoutcandidates were verified via PCR and Southern blot analysis todemonstrate gene deletion as well as loss of the kanamycin cassette.

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure that follows, the following abbreviationsapply: MOPS (3-[N-morpholino] propanesulfonic acid); PCR (polymerasechain reaction); vpB (vitelline protein B).

Example I

The studies described herein focus on highly attenuated mutants in orderto provide the highest degree of safety. Despite multiple hits in thesame genes, sequencing in progress has identified more than 100 distinctloci that contribute to intracellular survival. Double mutants have beenconstructed in a number of the most attenuated mutants. Double mutantsmay include two genes in the same metabolic pathway at separate geneticloci to virtually eliminate the potential for reversion of the vaccinestrain to virulence or distinctly different functions which whencombined may be expected to severely cripple the organism or provide adiagnostic target in order to distinguish vaccine from virulent fieldstrain. Transposon mutagenesis has identified several hundred Brucellagenes whose activity contributes to intracellular survival as providedfor in Ficht et al. (2000) Infection and Immunity 68, 102-107,incorporated herein by reference. Nonpolar knockout mutations have beenconstructed in a number of genetic loci identified as important forsurvival and protection induced by these strains against challenge wasexamined at several times post clearance. DNA flanking the internaldeletion fragment was amplified by PCR using primers designed withspecific restriction sites to create a nonpolar deletion and bothfragments were cloned into pBluescript or any other ColE1-based vectorthat cannot replicate in Brucella. A kanamycin resistance cassette isinserted at the junction of the two fragments and the resulting plasmidis electroporated into the parental Brucella strain. Marked-knockoutswere created by allelic exchange and selected based on their kanamycinresistance and ampicillin-sensitivity and were considered polar. Theseorganisms are then subjected to a second electroporation using a plasmidthat contains the same construct lacking the kanamycin cassette, butalso has the sacB and ampicillin-resistance genes. Cointegrants wereselected for carbenicillin resistance and sucrose sensitivity. This stepassures the integration of a functional sacB gene necessary for thefinal step. These colonies are incubated in media containing sucrose andplated onto sucrose plates. Individual colonies are selected and replicaplated onto sucrose plates and verified for kanamycin sensitivity andsucrose resistance. Verification of nonpolar knockout was performed byPCR, Southern blotting, and DNA sequencing.

In surveying the large numbers of transposon mutants developed, sequencedata has provided a great deal of information concerning the environmentin which Brucella persists. In a recent screen of mariner mutants, 40/42of the genes identified to date possess homology to entries in GenBank.Of these mutants, 31/42 were either known virulence factors or hadhomology to virulence factors described in other organisms, 6/42 werepurine auxotrophs, 10/42 were virB genes, and 2/42 possessed nosimilarity to entries in the database. Based on the preliminary resultsat least 3% of the Brucella genome appears to be required forintracellular survival. Although, screening in the mouse model suggestedat least a ten-fold higher number, it became too difficult to sortthrough mutants exhibiting lesser degrees of attenuation. It may beexpected that attenuated mutants unable to persist within macrophageswill fall into several different categories. Mutants exhibiting thegreatest level of attenuation and are therefore safest will be theprimary focus of this proposal. Genes involved in purine and glutamate(or nitrogen) metabolism have been identified within this group. Bothprovide a number of potential targets for elimination along complexpathways. Another group may be impaired in response to stressconditions, including those unable to withstand the bactericidalmechanisms of phagocytes such as the oxidative burst, acidity of thephagolysosome, or activity of defensins. A third group may have defectsin effector molecule secretion for example by the Brucella homolog tothe type IV secretion system. While not limited the present invention toany particular theory, it is possible that these molecules function to“modify the phagosome” and enhance survival or the equivalent oficm/dot. However, the inventors have also identified a number of novelclasses of genes that will have to be fully characterized in order toestablish their role in infection. Many in this final group may bedefective in specific host adaptations, e.g. adhesion to host cells,invasion, intracellular replication, systemic spread, or modulation ofthe host immune response.

One embodiment of the present invention involves the use of mutations inrecognized Brucella virulence factors to develop vaccines. Both singleand double mutations have been investigated to provide a balance betweensafety and persistence. While double knockouts likely provide thehighest degree of safety, some of these mutants will be too highlyattenuated to stimulate protective immunity even in encapsulated form ormay be missing important protective antigens. For purposes of thisproposal, the present inventors utilized knockouts whose identity andputative function are known. These include ΔvirB2 (type IV secretionsystem) and ΔmanB/A (LPS synthesis). Efficacy of these single mutantstrains alone is illustrated here (FIG. 1); vaccine potential ofindividual mutants was evaluated following clearance of vaccinecandidates (i.e., nonrecoverable) in order to minimize the contributionof non-specific immune functions to clearance.

B. melitensis Rev-1 (vaccine strain) was also used as a control in theprotection experiment. When considering clearance of these organismsfollowing vaccination, manB/A (ΔMBA), virB (ΔVB2) exhibit early, rapiddeclines in recovery (Group 1 classification, highly attenuated), whilethe ΔASP (Group 2) mutant exhibits a more gradual decline in number; thedouble mutant virB/manB/A exhibits the most rapid clearing time. Theresults observed (FIG. 1) appear to confirm the contention thatincreased protection is associated with persistence of vaccine strains.However, it is difficult to generalize since genes missing from themutants may also be important protective immunogens. The ΔVB2 shown hereand the ΔVB2/ΔMBA double mutant may be used effectively in oneembodiment of the present invention. The efficacy and persistence of theorganisms introduced in unencapsulated or encapsulated (controlledrelease) format may further be evaluated. The inventors hypothesize thatmaximal safety may be through use of the rapidly clearing strains, whileenhancing efficacy through the disclosed controlled release format.

The use of a novel biopolymer, vpB, in microcapsule formulations greatlyextends the time frame of erosion and release of capsule contents. Thisproperty is based on the unusual enzymatic and chemical resistance ofthe protein to breakdown. To demonstrate the unusual properties of thisprotein additive with regard to vaccination, the present inventors haveincluded it in both a solid microparticle and a reservoir microparticleformulation (alginate, FIG. 2), and monitored the enhancement of theimmune response in each case. Our methods include the addition of vpB(recombinant) derived from aquatic invertebrates, a protein of 31 kDaproduced in E. coli free of additional amino acids and purified throughconventional chromatography. The protein is non-antigenic and has beenextensively characterized regarding biochemical properties. We havedemonstrated the ability to form a variety of capsules with thismaterial altering the surface properties and controlled release profilesof the particles (data not shown). The performance of vpB containingreservoir capsules in vaccine enhancement is shown in FIG. 6. Armed withthe information that the vpB protein imparted sustained release profilesto erodible capsules, the inventors used this protein as an additive toextend release profiles of a number of capsule types. Alginate capsulesin particular were a target of interest as a hydrogel to stabilizeentrapped vaccine strains and maintain viability. Through a modificationof the methods of Abraham et al. (1996) Pharm. Dev. Technol. 1, 63-68,hereby incorporated by reference, the present inventors produced threeformulations of alginate-based microcapsules to entrap and deliver liveBrucella vaccines. Alginate solutions (1.5% in MOPS buffer) containing1×10⁶ bacteria/ml were nebulized into a solution of 100 mM CaCl₂ usingan Encapsulator with a 200 micron nozzle (Innova, Inc.) and stirred for15 minutes at 20° C. CaCl₂ solution was removed and crosslinking ofalginate achieved with a solution of 0.5% poly-L-lysine. An externalcoating of alginate was applied through a five-minute incubation ofparticles in a 0.03% (w/v) alginate solution in MOPS buffer. Particleswere washed with MOPS buffer before storage at 4° C. (formulation A). Asecond formulation employed the substitution of vpB for poly-L-lysine inthe crosslinking reaction, producing a vpB layer in the capsule wall(formulation C). A third formulation incorporated vpB (0.5 mg/ml) intothe alginate core with the bacterial payload to extend bacterial releasefrom the capsule and delay breakdown of the capsule (formulation B).Formulations A, B, and C were compared with unencapsulated bacteria,empty capsule type C and buffer for protection against challenge 32weeks after a single vaccination dose as shown in FIG. 6. Each of threecapsule formulations described above were tested in mice (FIG. 6)through intraperitoneal injection to perform as a depot. The vaccinestrain used 19C6 (ΔvjbR; BMEI1116) is one of the Group 1 attenuatedstrains discovered through transposon mutagenesis of B. melitensis. Thishas been revealed by screening attenuated strains in macrophage cultureand mice. In this study 1×10⁶ bacteria 19C6 were introduced into mice(n=10) intraperitoneally (IP). The six groups of animals received bufferalone (MOPS), empty capsules (alginate), unencapsulated 19C6 or one ofthree encapsulated forms of 19C6. The three forms include formulation A(alginate only), formulation B (alginate, vpB core) and formulation C(alginate, vpB shell). Thirty-two weeks following vaccination, allgroups were challenged with 1×10⁴ B. melitensis strain 16M through IPinjection. Vaccination of mice with the 19C6 strain alone provided threelogs of protection against challenge. Vaccination with the same organismin an encapsulated form provided one to two logs of additionalprotection. Formulation C, which provided the best protection, isformulated with a vpB shell; 50% of the animals in this groupdemonstrated sterile immunity after challenge, thirty-two weeksfollowing vaccination.

This study was carried out once with 60 mice and clearly demonstratesthe utility of encapsulation in immune potentiation. The inventors usedtwo highly attenuated strains for the studies described here. Thepresent inventors determined the clearing time for encapsulated Brucellafrom the mouse spleen following vaccination as a measure of safety (FIG.3). The present inventors have further tested a single mutant ΔVB and adouble mutant, ΔVB/ΔMBA for persistence and efficacy in an encapsulatedformat.

FIG. 4 shows recovery of B. melitensis 16M by bacteriologic culture fromspleen in BALB/c mice vaccinated with encapsulated B. melitensis vaccinecompositions of the present invention following 32 weekspost-vaccination. A single asterisk (*) represents data with p<0.05;double asterisks (**) represent data with p<0.01.

In related studies the inventors used cell fractionation and ELIspotanalysis to define the T cell subpopulations producing the observedcytokines and have provided an ELISA-based Bioplex analysis here toillustrate an example of data collected to date on the experimentalvaccination detailed in FIG. 6. Both humoral and cell mediated immunitywere followed during the course of the study. Prior to challenge, fivemice were evaluated for cell mediated immune status through splenocyteblastogenesis and release of cytokines into the supernatant (FIG. 6.).Following challenge the remaining mice were bled at 48 hours and serumevaluated for cytokine production. This same group was sacrificed at twoweeks post challenge and an enumeration of bacteria in the spleen wasused as an indicator of protection (FIG. 4). To summarize the analysisof immune parameters, the inventors found that serum antibody titerscorrelated with protection while blastogenesis results were unrevealing.Cytokine analysis of antigen-stimulated splenocytes reveals thehallmarks of a Th1 response in animals analyzed prior to challenge (FIG.6). Bioplex analysis of cytokines present in sera revealed similarprofiles. We will analyze T cell subpopulations via ELIspot in futureexperiments to gain a better understanding of immune correlates.

Cytokine quantitation: Spleens were excised from vaccinated mice andground lightly with the frosted ends of two glass slides. Erythrocyteswere lysed using ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 100 mMNa₂EDTA, pH 7.2-7.4) and the cell suspension was washed followingcentrifugation three times in RPMI-1640 medium. Then the concentrationof cells was adjusted to 2×10⁶ cells/ml with complete RPMI-1640containing 25 mM HEPES, 2 mM L-glutamine, 10% (v/v) heat-inactivated(for 30 min at 56° C.) fetal bovine serum (Difco), and 5.5×10⁵ M2-mercaptoethanol in the presence of 100 U penicillin and 10 mgstreptomycin (Difco). For cytokine production by splenocytes, 4×10⁶cells are cultured as described in a 24-well tissue culture treatedplate (Costar, Massachusetts) for three days at 37° C., 5% CO₂. 1×10⁸heat-killed B. melitensis 16M, or 1 μg of lysate is applied to eachwell. Cells in control wells receive complete RPMI-1640 only asbackground control. Cell suspensions are harvested and assayed forcytokine levels. Filtrates were analyzed by ELISA using Bio-Plexanalysis, a multiplex suspension array technique that relies on antigencapture and bioluminecence (Luminex Corp.) for IL-2, IL-4 and IL-10.Spleen supernatants were treated according to manufacturer'sinstructions. Sera were serially diluted and similarly analyzed. FIG. 6shows the results of ELISA analysis via Bioplex of IFN gamma in spleensupernatants from animals at 32 weeks post-vaccination. FIG. 5illustrates IFN gamma production detected in sera of animals at 10 and30 weeks post-vaccination and two days after challenge doses weredelivered. The relative levels of cytokines concur between theseanalyses although the levels of cytokine are much higher in spleensupernatants than in sera, as expected. Data has also been collected onIL-2, IL-4 and IL-10 revealing a Th1 cytokine profile elevated in IL2and IFN gamma and low in IL4 production. One may further use ELIspotanalysis to assign these values to specific T cell subsets.

Example II

The present inventors have previously used signature-tagged mutagenesisto identify in vitro and in vivo virulence genes (1, 16, 40). Amongthese, a mucR (BMEI 1364) mutant, was attenuated for survival in themouse and macrophage model (40). The role of MucR in Brucella isunknown, but recently, the function of the MucR gene has been identifiedin soil and plant bacteria such as Agrobacterium tumefaciencis andSinorhizobium meliloti (19, 28). MucR is a transcriptional regulatorthat coordinates a diverse set of bacterial behaviors including thecontrol of exopolysaccharide production which is important not only inbacterial-plant symbiosis but also in biofilm formation(4, 5).

In the present invention the inventors conducted a series of studiesdesigned to characterize the Brucella melitensis 16MΔmucR as a potentialvaccine candidate against intraperitoneal and aerosol Brucellamelitensis 16M challenge. Vaccination with the mutant did not inducesystemic or local adverse reactions and significantly protected BALB/cmice against intraperitoneal and aerosol challenge.

Materials and Methods: (i) Mice: 6 to 8-week old female BALB/c mice wereobtained from the Jackson Laboratories (Bar Harbor, Me.) and acclimatedfor one week prior to infection or vaccination. All experimentalprocedures and animal care were performed in compliance with theinstitutional animal care guidelines.

Bacterial strains: Strains used in these studies include B. melitensis16MΔmucR (engineered for this study and used as the vaccine candidate)and the virulent strain Brucella melitensis 16M biovar 1 (originallyobtained from ATCC and re-isolated by this lab from an aborted goatfetus) (24). Bacteria were grown on tryptic soy agar (TSA) (Difco,Becton Dickinson) or Farrell's media (TSA supplemented with OxoidBrucella supplement) at 37° C. with 5% CO₂. For construction of the B.melitensis 16MΔmucR knockout, the medium was supplemented with kanamycin(100 μg/ml), or carbenicillin (100 μg/ml). Sucrose media was utilizedfor sacB counterselection and unmarked gene deletion selection aspreviously described(25). Escherichia coli cultures utilized for theconstruction of the 16MΔmucR mutant were grown on Luria-Bertani (LB)(Difco, Becton Dickinson) plates or in LB broth overnight at 37° C. withor without ampicillin (100 mg/liter) or kanamycin (100 mg/liter).

To prepare organism for animal infections, Brucella were harvested fromthe surface of the plates after 3 days of incubation usingphosphate-buffered saline (PBS) pH 7.2. The bacteria were pelleted andresuspended to a final concentration based on optical density readingsusing a Klett meter and a standardized curve. Actual viable counts wereconfirmed by serial dilution, plating, and enumeration.

Construction of the Brucella melitensis 16MΔmucR deletion mutant: Themutant was constructed as previously described with some modifications(25). The sequence upstream of the mucR gene (BMEI1364) was amplifiedfrom B. melitensis 16M with the primer pair5′GCTCTAGAGCCCATCAACAACAGGACAAACGG3′ (SEQ ID NO: 13) (contains XbaIsite) and 5′GGCGGCGCGCCTGGTTGCTCCGAACTATGCTG (SEQ ID NO: 14) (containsAscI site). The sequence downstream of mucR was amplified with theprimer pair 5′CCAGGCGCGCCGCCGCTGCGTATTTCATAATC (SEQ ID NO: 15) (containsAscI site) and 5′GCTCTAGAGCCTTTGCAGGTTTTCCGTATCTTT (SEQ ID NO: 16)(contains XbaI site). These two products were ligated to one another viaoverlapping PCR via the AscI site (New England Biolabs) engineeredbetween the two sequences. This overlap product was then ligated topEX18Ap via the XbaI site (named pMMK40). A kanamycin resistance genewas subsequently ligated within the vector by the unique AscI site(plasmid pMMK44). This construct was used for electroporation intoBrucella melitensis 16M. Potential marked deletion mutants werekanamycin resistant and carbenicillin sensitive, and were verified byPCR and Southern Blot; the confirmed mutant was named 16MΔmucR::Kan. Theunmarked deletion mutant was engineered by electroporation of pMMK40into 16MΔmucR::Kan and selected on TSA/carb@100. Cointegrants with thefollowing phenotypes were selected: Kan^(R), Carb^(R), and sucrose^(S),indicating a co-integrant with a functional sacB gene. Bacteria wereselected in the presence of sucrose for resolution of co-integration aspreviously described(25). All knockout candidates were verified by PCRand Southern Blot to demonstrate gene deletion and loss of the kanamycincassette.

Evaluation of B. melitensis 16MΔmucR attenuation in mice: Forty, 6-8week old female BALB/c mice were used to evaluate the persistence andreplication of the B. melitensis 16MΔmucR mutant. Mice were inoculatedintraperitoneally with either (a) 1×10⁶ CFU in 100 μl 16MΔmucR or (b)1×10⁶ CFU in 100 μl of the parental strain 16M. Groups of four mice wereeuthanized via carbon dioxide asphyxiation at 1, 3, 5, 7 or 9 weeks postinfection. At each time point, the spleens were harvested, weighed, andhomogenized in 1 ml of peptone saline. Serial dilutions were prepared,and 100 μl aliquots of each dilution (including the undiluted organ)were plated in duplicate onto TSA plates. The levels of infection wereexpressed as the mean±of standard error of the mean (SEM) of individuallog CFU/spleen (FIG. 7A). Elevated spleen weights indicative ofsplenomegaly in the 16M parental strain were not not observed in thevaccine strain (FIG. 7B).

Immunization of mice for efficacy studies: Six to eight week-old femaleBALB/c mice were distributed into 3 treatment groups and inoculatedintraperitoneally (IP) with a single dose of B. melitensis 16MΔmucR.Treatment groups included: (a) 1×10⁵ CFU/mouse, (b) 1×10⁶ CFU/mouse, or(c) PBS as a control. Mice were housed for 20 weeks post-vaccinationunder ABSL-3 conditions.

Vaccination efficacy against intraperitoneal challenge: Twenty weekspost-vaccination, mice (n=5 per group) were challenged i.p using 6×10⁵CFU/mouse of B. melitensis 16M. One week post-challenge, mice wereeuthanized via CO₂ asphyxiation. Spleens, lungs, and livers werecollected, weighed and homogenized in 1 ml of PBS. Serial dilutions wereperformed and aliquots were plated onto TSA or Farrell's media plates.One week post-challenge, animals were euthanized, spleens, lungs andlivers harvested, homogenized and plated to determine total CFU/organ.The levels of infection were expressed as means±SEMs of the individuallog₁₀ CFU/spleen, log₁₀ CFU/liver and log₁₀ CFU/lung (FIG. 8).

Vaccination efficacy against aerosol challenge: Twenty weeks postvaccination, groups of 5 mice were challenged with an aerosol chamberdose of 5×10⁹ CFU/ml of B. melitensis 16M. Four weeks post challenge,the mice were euthanized and the lungs, liver and spleen were removed,weighed, homogenized in 1 ml PBS, serially diluted and plated ontoFarrell's medium to determine total CFU/organ. The levels of infectionwere expressed as means±of SEMs of the individual log₁₀ CFU/spleen,log₁₀ CFU/liver and log₁₀ CFU/lung. A group of 3 mice were euthanizeddirectly after aerosol exposure to quantify the CFU/lung inhaled (FIG.9).

Cross protection against other species of Brucella: Six to eightweek-old female BALB/c mice are inoculated intraperitoneally (IP) with asingle dose (1×10⁶ CFU/mouse) of B. melitensis 16MΔVirB,16MΔVirB/ManB/A, 16MΔvjbR. Controls include empty capsules or PBS as acontrol. Twenty weeks post-vaccination, mice (n=5 per group) werechallenged i.p using 1×10⁴ CFU/mouse of B. melitensis 16M or B. abortus2308. One week post-challenge, mice were euthanized via CO₂asphyxiation. Spleens, lungs, and livers were collected, weighed andhomogenized in lml of PBS. Serial dilutions were performed and aliquotswere plated onto TSA or Farrell's media plates. One week post-challenge,animals were euthanized, spleens, lungs and livers harvested,homogenized and plated to determine total CFU/organ. The levels ofinfection were expressed as means±SEMs of the individual log₁₀CFU/spleen, log₁₀ CFU/liver and log₁₀ CFU/lung. Cross protection wasobserved for encapsulated B. melitensis ΔvirB2 and for encapsulated B.melitensis ΔvjbR (FIGS. 10A and 10B).

Safety Studies with vjbR mutants in B. melitensis, B. abortus S19:IRF-/IRF-(interferon regulatory gene) knockout mice are considered agood model for immunocompromised subjects. IRF-1^(−/−) mice wereinfected intraperitoneally with 1×10⁶ CFU/mouse of either B. melitensis16MΔvjbR, B. abortus S19ΔvjbR, B. melitensis 16M, B. abortus 2308 or S19(FIG. 11). Mice inoculated with ΔvjbR vaccine candidates survived longercompared to either 16M (P<0.005), 2308 (P<0.005) or S19 (P<0.005) In thepresent study, the safety of the vaccine candidates in the Interferonregulatory factor (IRF^(−/−)) knockout mice. IRF-1^(−/−) mice infectedwith either wild-type Brucella melitensis 16M or the vaccine strainBrucella abortus S19, succumb to the disease within the first threeweeks of infection, which is characterized by a marked granulomatous andneutrophilic inflammatory response that principally targets the spleenand liver. In contrast, IRF-1^(−/−) mice inoculated with either the B.melitensis 16MΔvjbR or B. abortus S19ΔvjbR, do not show any clinical ormajor pathologic changes associated with vaccination. Additionally, when16MΔvjbR or S19ΔvjbR vaccinated mice are challenged with wild-typeBrucella melitensis 16M, the degree of colonization in multiple organsis significantly reduced, along with associated pathologic changes.These findings demonstrate the safety and protective efficacy of thevjbR mutant in an immunocompromised mouse model.

Histopathology: Twelve female BALB/c mice were distributed into 4groups. The groups consisted of A) 16MΔmucR vaccinated and subsequentlyaerosol challenged (2 weeks post-challenge) B and C) Non vaccinated(naïve) and aerosol challenged (2 and 4 weeks post-challenge) and D) nonvaccinated and IP challenged (4 weeks post-challenge). Animals wereeuthanized by CO₂ asphyxiation, and spleen, lungs, liver, kidneys andheart were harvested, fixed in 10% buffered formalin, paraffin embeddedand stained with hematoxylin and eosin. Histological changes wereassessed between treatment groups (FIGS. 12A-12D, and FIGS. 13A-13D).

Statistical analysis: Bacterial burden from mutant clearance as well asefficacy studies were expressed as mean CFU+/−standard error (SE) andpresented graphically as the log₁₀ CFU Brucella recovered per organ.Culture-negative organs were assigned a value of four CFU, which isbelow the limit of detection of five CFU/organ. Spleen weight data fromkinetics was plotted as the mean spleen weight in mg+/−standard error(SE).

For the survival of 16MΔmucR in mice, a Student's T-test was performedto compare splenic colonization and weight of the knockout strain to thewildtype control group at each timepoint (FIGS. 7A and 7B). Efficacystudies compared vaccinated and subsequently challenged mice to micereceiving PBS as a vaccine control that were challenged with wild typeorganism. In the intraperitoneal challenge study, statisticalsignificance of differences between vaccinates were analyzed by ANOVAfor each organ separately followed by Tukey's honestly significant (HSD)post-test comparing all groups to one another (FIG. 8). In the aerosolprotection studies a Student's T-test was performed for each organseparately to compare the vaccines to naïve mice. For all statisticalanalyses P-values less than 0.05 were considered statisticallysignificant (FIG. 9).

Attenuation of 16MΔmucR in mice: To determine the effect of the mucRgene deletion in vivo, mice were infected IP with 1×10⁶ CFU/mouse of B.melitensis 16MΔmucR. Compared to the wild type strain 16M, thecolonization of the spleen with 16MΔmucR was significantly reduced at 1,3 and 6 weeks (P<0.01), but not at 9 or 12 weeks (FIG. 7A). Reducedsplenic colonization by the 16MΔmucR mutant correlated with reducedspleen weights (FIG. 7B), indicating a reduced inflammatory response bythe mutant. Spleen weights of mice infected with wild-type 16M wereconsistently higher.

Evaluation of immune protection provided by 16MΔmucR againstintraperitoneal 16M challenge: In order to determine the vaccinationefficacy elicited by the 16MΔmucR mutant, the level of protectionprovided by the vaccine candidate was evaluated against intraperitonealB. melitensis 16M wild-type challenge at 20 weeks postvaccination.Animals were euthanized one week post-challenge because this timepointcorresponds to the highest bacterial load in the spleen based onprevious studies (25). At one week post challenge (21 weekspostvaccination), there was a statistically significant decrease in thesplenic, hepatic and pulmonary bacterial loads from the mice vaccinatedwith the 16MΔmucR mutant relative to those of the naïve mice regardlessof the vaccination dose, with a 4.14-4.75 log reduction in bacterialburden in the spleen (P<0.001), 3.24-3.34 log reduction in the liver(P<0.001) and 2.54-3.64 log reduction in the lungs (P<0.001) (FIG. 8).

Evaluation of immune protection provided by 16ΔmucR against aerosol 16Mchallenge: In order to determine the vaccination efficacy elicited bythe 16MΔmucR mutant against a natural route of exposure, the level ofprotection provided by the vaccine candidate was evaluated againstaerosol B. melitensis 16M wild-type challenge at 20 weekspostvaccination. For aerosol exposure studies, the four weekpost-challenge timepoint was chosen as the timepoint of peak spleniccolonization(23). Mice that were euthanized within 1 hour of aerosolexposure inhaled an average of 2.1×10⁴ CFU/lungs as determined byplating their lungs and enumerating the bacteria recovered.

When mice were challenged by the aerosol route, the 16MΔmucR vaccinecandidate protected mice significantly in all organs plated. The mutantafforded a 2.79 log reduction in the bacterial burden in the spleen(P<0.05), 1.97 log reduction in the liver (P<0.05) and a 1.63 logreduction in the lungs (P<0.01) (FIG. 9).

Vaccination of mice with B. melitensis ΔvjbR strain protects against B.abortus challenge as well as B. melitensis challenge. There should begood cross protection whether a B. abortus or a B. melitensis strain isused as vaccine based on previously described data. Exception isΔvirB/ΔManB (FIGS. 10A and 10B).

Survival of the Brucella ΔvjbR mutants in IRF-1^(−/−) knockout mice: Tendays post inoculation, 60% of mice had succumbed to the infection withB. melitensis 16M (FIG. 11). Similarly, mice inoculated with B. abortus2308 exhibited signs of illness by day 7 and 50% of mice were euthanizedby day 11 post-inoculation due to imminent deterioration (FIG. 11).Interestingly, mice vaccinated with the vaccine strain S19, alsoelicited clinical signs of illness, but at a later timepoint thanobserved with the virulent strains, i.e., at day 10 post-inoculationwith a 100% mortality rate by day 28 (FIG. 11). In contrast, 100% ofmice vaccinated with either the 16MΔvjbR or S19ΔvjbR mutant exhibited nosigns of disease (P<0.0001) and 100% survived beyond day 30post-inoculation (FIG. 11). Two animals from the B. melitensis 16M andB. abortus 2308 exhibited no signs of disease beyond day 30post-inoculation. This data clearly demonstrate that vjbR mutants areless virulent in immunocompromised mice than the parental strains fromwhich they were derived.

Microscopic changes observed in the spleens (FIGS. 12A-12D) and in thelivers (FIGS. 13A-13D) of IRF-1^(−/−) mice vaccinated with S19ΔvjbR(FIGS. 12C and 13C) or 16MΔvjbR (FIGS. 12D and 13D) and challenged 8weeks post-vaccination with 1×10⁶ CFU/mouse of wild-type B. melitensis16M. Naïve but challenged mice (FIGS. 12B and 13B) or naïve (FIGS. 12Aand 13A) are presented for comparison. It can be seen from the figuresthat there is a marked reduction in the inflammatory response in boththe spleen and liver in animals that received the ΔvjbR mutants.

Evaluation of histological changes in mice vaccinated with 16MΔmucR:Histological analysis of the lungs, livers, and spleens of BALB/c miceinoculated with either 16M_mucR (FIGS. 14D-14F) or naive PBS controls(FIGS. 14A-14C) that were subsequently aerosol challenged with wild-type16M and euthanized 2 weeks postchallenge was assessed to determine thedegree of inflammation elicited by the challenge organism in animalsthat were vaccinated with the mutant. Histologically, thebronchiole-associated lymphoid tissue was prominent at 2 weekspostchallenge (FIG. 14A), and no significant changes were seen in micevaccinated with the mucR mutant (FIG. 14D). Inflammatory foci in theliver of naive aerosol challenged mice were rare at 2 weekspostchallenge (FIG. 14B) and absent in mice vaccinated with the mucRmutant (FIG. 14E). Changes in the spleen of naive mice consisted ofmarked extramedullary hematopoiesis at 2 weeks postchallenge (FIG. 14C).There were no significant changes in the spleen of mice vaccinated withthe mucR mutant (FIG. 14F). No significant changes were observed in thekidneys or hearts in vaccinated or aerosol exposed animals (data notshown). Naive mice are depicted in FIGS. 14G-14I for comparison.

Historically, the most efficacious vaccines against brucellosis havebeen live attenuated vaccines, and is the case of multiple currentlylicensed vaccine strains for animal use including S19, Rev 1 andRB51(15, 37). Unfortunately, both S19 and Rev 1 vaccines, which havebeen highly efficacious in controlling the disease in cattle and goatsrespectively, have proven to be unsafe or have the capacity to causeadverse reactions in humans due to local and systemic reactions that insome cases resulted in the development of the disease (3, 38). Othervaccinology alternatives including the use of subunit, recombinantproteins and DNA vaccines which might be safer for human use, althoughcapable of eliciting both humoral and cellular immune responses to acertain degree, generally induce lower or no protection compared to thelive attenuated vaccines in animal models (8-10, 14, 30, 32, 33, 36). Assuch, a live attenuated organism has been utilized as the vaccine typeof choice for the prevention of Brucellosis. An ideal Brucella vaccinewould be one that persists long enough to generate a robust immuneresponse without eliciting the undesired side effects such assplenomegaly or clinical signs of disease.

Previous studies using signature tagged mutagenesis by the presentinventors have identified multiple candidate genes that are attenuatedfor virulence and survival in the mouse and macrophage models, amongthese disruption of the mucR locus in B. melitensis 16M(40). Theinventors have previously demonstrated an in vitro and in vivo role forthe mucR transposon mutant. The organism was found to be significantlyattenuated in both models when the gene was interrupted. To furthercharacterize the role of mucR in regards to survival, protectiveefficacy and safety in vivo, an unmarked gene deletion was created.

In Sinorhizobium meliloti, a gram-negative soil bacterium thatestablishes a symbiotic relationship with alfalfa, a clear role of themucR gene has been recently established(28). MucR has been identified asa transcriptional regulator with multiple functions that help in theestablishment of symbiosis, including a key role in the control ofexopolysaccharide biosynthesis, necessary for biofilm formation(4, 5,35). Biofilms are microbial aggregates surrounded by a self-producedmatrix that attach to a surface (11, 12). The biofilm provides bacteriawith a physical barrier against antibiotics, innate defense mechanismsfrom the host and environmental stress conditions including UVradiation, pH changes and osmotic shock among others (11). Importantcomponents of biofilms include water, bacterial cells andexopolysaccharides(11). Exopolysaccharides have been recognized as keyelements that provide the structural support for the biofilm. In orderto ensure a successful symbiotic association, exopolysaccharideproduction and biofilm formation are tightly regulated and partiallycontrolled by the mucR gene. Deletion of mucR in S. meliloti thereforeresults in deficiencies in invasion or the establishment of symbiosis.

One clear example of the importance of the mucR gene in S. melilotisymbiosis is in the induction of nodule formation(28). MucR causes anincrease in the biosynthesis of nod factor, necessary for the inductionof nodule development. Other established roles of mucR in this organisminclude an induction of increased expression of multiple operonsrequired for nitrogen fixation and respiration, as well as numerous typeIV secretion systems and putative transport-related genes all necessaryfor a successful symbiosis(28).

In the case of Brucella, the role of mucR is less understood.Preliminary studies from this laboratory using microarray technologysuggests that the mucR gene regulates exopolysaccharide biosynthesis, aswell as genes involved in iron sequestration and storage, nitrogenmetabolism and stress response mechanisms. (J. Weeks, unpublished data).Although preliminary and still under investigation, all these putativeroles of mucR in Brucella explain to a certain degree the attenuation ofthe mutant strain observed in J774A macrophages and in mice. Recently ithas been reported that B. melitensis 16M produces an exopolysaccharide;studies suggested that Brucella may indeed be capable of biofilmformation (17). It is possible that mucR may play a role in biofilmformation through regulation of exopolysaccharide synthesis.

Protective efficacy as a function of persistence has been previouslyevaluated by this laboratory(25). The construction and characterizationof multiple deletion mutants in Brucella abortus and Brucella melitensishas led to the conclusion that a vaccine candidate needs to persist inthe host long enough in order to mount a strong protective immuneresponse(15, 25). This observation is apparent here as well with the B.melitensis mucR mutant. Interestingly, the mucR mutant persists at leastfor 12 weeks in mice, similarly to the wild-type 16M, but the degree ofcolonization is significantly reduced compared to the parental strainduring the acute phase of the infection. This difference in colonizationproperties may explain the lack of gross and microscopic changesassociated with infection. Lack of hepatic granuloma formation,hepatomegaly or splenomegaly associated with vaccination suggests thatimmunization with the mutant is safe, and therefore superior to manyother Brucella vaccines, including licensed ones. Most importantly,protection against the most common microscopic changes associated withthe disease in mice such as granulomatous hepatitis, granulomatoussplenitis or splenomegaly was not observed, indicating that vaccinationwith the mucR mutant not only reduced the bacterial burden in multipleorgans but also prevents against the development of Brucella-associatedpathologic changes. Lack of splenomegaly associated with vaccination hasbeen previously demonstrated as a safety parameter in other vaccinecandidates (2).

Protection against intraperitoneal challenge, observing the output ofbacterial colonization in the spleen of mice, has been historically usedas a means of evaluating Brucella vaccine efficacy (6, 27, 34, 39).Although this vaccination or challenge location does not reflect anatural route of infection, it has been extremely useful in determiningthe potential efficacy of vaccine candidates against brucellosis. Mostimportantly, it provides a reproducible and invariable means ofcomparing multiple vaccine candidate strains that had been studied forthe past 30 to 50 years. When 16MΔmucR vaccinated mice were challengedagainst wild-type Brucella melitensis 16M, all animals demonstrated astatistically significant reduction in the bacterial burden in thespleen, lung and liver regardless of the vaccination dose. The markedreduction in bacterial burden in the spleen conferred by the mutant isimpressive and comparable to other live attenuated vaccine candidatestested by this laboratory and others(2, 20, 21, 25). Although anintraperitoneal challenge is of historical importance, a more logicalapproach is the use of an aerosol challenge route, not only because ofthe documented evidence of aerosol transmission of these organisms, butalso because of the potential threat of the use of Brucella as abioterrorism agent(13, 18, 22, 26). It has been documented that 10 to100 organisms are enough to cause disease in humans, and Brucella istherefore considered highly infectious when delivered by this route (7).Previous investigations performed by this laboratory has determined thatBALB/c mice receiving an infectious dose of 5×109 CFU/ml added to thechamber nebulizer inhaled an average of 12,250 organisms per mouse (4.10logs) and that tissue colonization reached a peak by 4 weekspost-exposure(23). The high dose (100-fold more bacteria that actuallyneeded to establish an infection) and the time post-vaccination chosento test efficacy, provide us the means to evaluate the vaccine candidateefficacy at the most stringent conditions. Interestingly andimportantly, the bacterial burden in the spleen, liver and lung weremarkedly reduced in animals that received the vaccine, demonstrating thevaccine efficacy against an aerosol exposure. Gross and microscopicevaluation confirmed the protection against the pathologic changesassociated with the disease. As expected, the highest number ofbacterial were isolated from the lung. It is possible that a diminishedinflammatory response in the lungs masks the efficacy in reducing thebacterial colonization, and although high bacterial counts were observedthere were no significant gross or microscopic changes associated withthe infection apart from an increased amount of BALT associated lymphoidtissue.

In the present invention, the intraperitoneal vaccination with the liveattenuated vaccine candidate 16MΔmucR was able to markedly enhance thebacterial clearance in the spleen, lung and liver using two differentchallenge routes. Most importantly, vaccination conferred protectionagainst Brucella-associated pathologic changes.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It may be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it may beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method for prophylaxis, immunization,amelioration of symptoms, or any combinations thereof againstbrucellosis in a human or animal subject comprising the steps of:identifying the human or animal subject in need of the prophylaxis,immunization, amelioration of symptoms, or any combinations thereofagainst brucellosis; and administering a therapeutically effectiveamount of a vaccine composition to the human or animal subject for theprophylaxis, amelioration of symptoms, or any combinations thereofagainst brucellosis, wherein the vaccine comprises: a Brucella straincomprising one or more attenuating gene knockouts and further comprisinga diagnostic gene knockout; an encapsulating agent comprising vitellineprotein B capable of releasing the Brucella strain at a predeterminedrate; and an optional adjuvant or a pharmaceutically acceptable carrier.2. The method of claim 1, wherein the Brucella strain is selected fromthe group consisting of Brucella melitensis and Brucella abortus.
 3. Themethod of claim 2, wherein the Brucella abortus is a Brucella abortusS19 strain.
 4. The method of claim 1, wherein the encapsulating agent isan alginate bead or a microsphere.
 5. The method of claim 1, wherein theattenuating gene knockout is selected from the group consisting ofΔvjbR, ΔmucR, ΔmanB/A, Δasp24, ΔvirB1, ΔvirB2, ΔvirB3, ΔvirB4, ΔvirB5,ΔvirB6, ΔvirB7, ΔvirB8, ΔvirB9, ΔvirB10, and ΔvirB11.
 6. The method ofclaim 1, wherein said diagnostic gene knockout comprises adifferentiation of infected animals from vaccinated animals (DIVA)mutant that includes ΔvirB12, Δbcsp31, and Δasp24.
 7. The method ofclaim 1, wherein the attenuating gene knockout comprises ΔvjbR, ΔmucR,ΔvirB2, or ΔmanB/A.
 8. The method of claim 1, wherein the vaccinefurther comprises a marker for serological testing, wherein the markeris a DIVA mutant comprising at least one of ΔvirB12, Δbcsp31, or Δasp24.9. The method of claim 1, wherein the vaccine comprises ΔmucR/DIVA,ΔvjbR/DIVA, ΔvirB2/DIVA, ΔmanB/A/DIVA, or any combinations thereof. 10.The method of claim 1, wherein the strain is a double mutant and furthercomprises a third mutation, wherein the third mutation is a marker forserological testing.
 11. The method of claim 10, wherein the doublemutant is selected from the group consisting of a ΔvirB2/ΔmanB/A mutant,ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant, ΔvirB2/ΔvjbR mutant,ΔvjbR/ΔmanB/A mutant, and ΔmucR/ΔmanB/A mutant.
 12. The method of claim1, wherein the strain comprises ΔvjbR/ΔmucR/DIVA mutant,ΔvirB2/ΔmanB/A/DIVA mutant, ΔvirB2/ΔvjbR/DIVA mutant, ΔvirB2/ΔmucR/DIVAmutant, ΔvjbR/ΔmanB/A/DIVA mutant, and ΔmucR/ΔmanB/A/DIVA mutant. 13.The method of claim 1, wherein the vaccine is administered by an oral,an intranasal, a parenteral, an intradermal, an intramuscular, anintraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal,a rectal, a vaginal, a sublingual, or a buccal route.
 14. A vaccinecomposition comprising: a Brucella strain comprising one or moreattenuating gene knockouts and further comprising a diagnostic geneknockout; and an encapsulating agent comprising vitelline protein Bcapable of releasing the Brucella strain at a predetermined rate.
 15. Amethod of making a vaccine against brucellosis in an animal subjectcomprising the steps of: providing a single or a double mutant strain ofBrucella comprising one or more attenuating gene knockouts and furthercomprising a diagnostic gene knockout; and an encapsulating agentcomprising vitelline protein B capable of releasing the Brucella strainat a predetermined rate.
 16. The method of claim 15, wherein theBrucella strain is selected from the group consisting of Brucellamelitensis and Brucella abortus.
 17. The method of claim 15, wherein theattenuating gene knockouts comprise ΔmucR, ΔvjbR, ΔvirB2, ΔmanB/A,ΔvirB2/ΔmanB/A mutant, ΔvjbR/ΔmucR mutant, ΔvirB2/ΔmucR mutant,ΔvirB2/ΔvjbR mutant, ΔvjbR/ΔmanB/A mutant, ΔmucR/ΔmanB/A mutant, or anycombinations thereof,
 18. The method of claim 15, wherein the diagnosticgene knockout comprises a differentiation of infected animals fromvaccinated animals (DIVA) mutant that includes at least one of ΔvirB12,Δbcsp31, Δasp24, or any combinations thereof
 19. The method of claim 16,wherein the Brucella abortus is a Brucella abortus S19 strain.
 20. Themethod of claim 15, wherein the vaccine is administered by an oral, anintranasal, a parenteral, an intradermal, an intramuscular, anintraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal,a rectal, a vaginal, a sublingual, or a buccal route.